Comparative microscopic observations of arbuscular mycorrhizal fungi after colonization of five Tunisian olive cultivars

1 Research Laboratory, Improving the Productivity of Olive Tree and Product Quality, Olive Tree Institute, 40 Rue Ibn Khouldoun, PB 14, 4061 Sousse, Tunisia. 2 Faculty of Science of Gabes, Rue Riadh, Zrig, 6072 Gabés, Tunisia. 3 Biochemical Laboratory, Research Laboratory LR12ES05: Lab-NAFS „Nutrition-Functional Food and Vascular Health‟, Faculty of Medicine, University of Monastir, Tunisia. 4 Agricultural Research, Agronutrition, 3 Avenue Orchid, Activestre Park, 31390 Carbonne, France.


INTRODUCTION
AMF is a beneficial form of symbiosis microorganism for majority of plants because of their positive effect on plant growth, rhizosphere volume (Al-Karaki, 2000) and a sustainable symbiosis with which to improve drought tolerance in horticultural crops, including fruit trees, vegetables and flowers (Jayne and Quigley, 2014;Baum et al., 2015).Bonfante and Genre (2015) described the "plantish" or "fungish" signals process as a hot topic in the field of plant-microbe interactions.However, plants and microbes exchange signals control each other"s development and metabolism and ultimately alter their interactions.
In general, lack of nutrients limits plant growth.Therefore, plants usually interact with microorganisms provided by their environment (Raaijmakers et al., 2009;López-Ráez et al., 2011).By this, they are able to establish symbiosis with some of microorganisms present in their rhizosphere.One of the most well studied beneficial plant-microorganism interactions is that established with Arbuscular Mycorrhizal Fungi (AMF).Smith and Read (2008) reported that this symbiotic association affects 95% of the plants.A good thing is that, AMF are well known in agriculture because they increase root surface area for water and nutrients absorption (Ortas and Ustuner, 2014).Infact, they enhance significantly, plant growth and tolerance, against biotic and abiotic stresses (Pozo and Azcón-Aguilar, 2007) and also soil texture (Smith, 1997).The mycorrhizal hyphae grow from roots to soil, producing roots which come in contact with additional area of soil thus increasing water absorption and nutrients uptake (Soriano-martı, 2009).
The olive (Olea europaea L.) is one of the oldest cultivated fruit trees in Tunisia, where it is grown traditionally in rain-fed conditions (Mechri et al., 2014).In order to stop the use of chemical fertilizers, there has been an increasing interest in the use of mycorrhiza in agricultural applications.Indeed, AMF interferes well with olive tree (Soriano-martı 2009), especially, Tunisian olive cultivars which showed the best compatibility with of Glomus species in enhancing production and growth (Khabou et al., 2014) and antioxidant activities by the accumulation of flavonoids and phenolic compounds (Mechri et al., 2015).Soriano-martı (2009) and Calvente et al. (2004) agreed that AM fungi induced significant increases in the growth, water relations, nutrient uptake, photosynthesis and tolerance to salinity in olive trees.Combination of AM and the Arbequina cv enhanced plant development and production while with Mission cv it increased root and leaf phenolic contents (Ganz et al., 2002).On the other hand, the inoculation of Glomus mosseae in Morailo and Frantoio cultivars increased plant growth, but did not show any significant difference for Leccino cv.When inoculated with G. intraradices, Arbequina cv showed higher development in terms of growth as compared to G. mosseae (Estaun et al., 2003).Study provided by Estaun et al. (2003) demonstrated that different olive cultivars have different responses to the same fungal species.Similarly, the same cultivar can respond differently to different fungal species.
In the present study, the main purpose was to obtain a general idea about AMF symbiosis established with olive plantlets of Tunisian cultivars.It aimed to compare microscopic observations of G. intraradices colonized olive roots of each local cultivar (cvs Oueslati, Meski, Jarboui, Chemlali and Chetoui) and to quantify the variation of mycorrhizal colonization.For this reason, focus was on the degree of M% and A% colonizations, of which no previous study has been carried out in Tunisia to examine the myccorhizal status for this case.

Experimental design
The experiment was carried out at the Olive institute of Sousse in Tunisia (35°50"N; 10°37"E).Olive (O.europaea L.) plantlets (15 cm long and three pairs of leaves) from cvs Oueslati, Meski, Jarboui, Chemlali and Chetoui were well watered and maintained in greenhouse.The air temperature of the greenhouse and relative humidity were about 25°C and 54%, respectively.After 2 weeks of acclimatization in a greenhouse, the olive plantlets were inoculated with "G.intraradices".1000 spores of G. intraradices were deposited directly below the roots of the olive plantlets (Mechri et al., 2014).Spores of "G.intraradices" used in this experiment were purchased from Agronutrition (France).Six months after planting, the plants were harvested and only roots of each cultivar were prepared for microscopic observations.Results were compared with uninoculated control plants for each cultivar.

Microscopic observations
The presence of an AMF infection was determined visually by clearing washed roots in 10% KOH and staining the preparation with 0.05% (vol/vol/vol) trypan blue in lactoglycerol and distilled water.According to the method of Trouvelot et al. (1986), the estimate of percentage of root colonization was calculated.The method involved putting five fragments of colored roots between strips in glycerol and then observing them in a light microscope.For each cultivar, six observations were prepared.Therefore, at least 30 replicates were used.Then, the observed fragments were noted in a grid based on a scale class.Calculation of the Intensity of mycorrhizal colonization in the root system (M%) and the Abundance of arbuscular in the root system (A%) were processed with the "Mycocalc" online program available freely from: http://www2.dijon.inra.fr/mychintec/.Photos of arbuscules and vesicles were taken with a digital camera coupled to the inverted microscope using "image J software".

Statistical analysis
Results were statistically analyzed using the SPSS statistical software version 18.0.The significance of differences between mean values was determined by one way analysis of variance.
Duncan"s test was used to compare the means.Differences *Corresponding author.E-mail: nawrazelmbarki@yahoo.com.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  between parameter means were considered significant when the P values of the ANOVA Duncan test were less than or equal to 0.05.

RESULTS AND DISCUSSION
This study highlighted olive root colonization by arbuscular fungi "G.intraradices" in Tunisia.Microscopic observations shown (Figure 1) revealed the presence of mycelium on one hand, and arbuscules (A) and vesicles (V), on the other.The "mycocalc" program was used to evaluate mycorrhizal colonization.Indeed, it is apparent that olive roots of Oueslati cv colonized by "G.intraradices" maintain higher colonization levels compared to the other four cultivars.Results presented in Figures 2 3 showed a significant difference in the percentage of root colonization (M%) and the arbuscules abundance (A%) between the five cultivars.Oueslati cv (50.47%) (Figure 2) has more colonization in terms of M%, compared to chetoui cv which has the lowest (41.90%).Similarly, the results showed higher percentage of Arbuscules abundance (A%) (Figure 3) in Oueslati cv (43.44%) compared to Meski (41.46%),Jarboui (41.40%)Chemlali (34.17%) and Chetoui (35.02%) cultivars and non-inoculated control plants.Wu and Zou (2010) reported that the AMF symbiosis established induces modifications in the root architecture of plants, in particular root length, diameter, density and the number of secondary roots.The present results demonstrated the dependence of the olive tree to arbuscular mycorrhizal fungi.Root colonization induced significant changes in the root structures of olive tree for Oueslati, Meski, Jarboui, Chemlali and Chetoui cultivars, Each value is the mean of three replicates ± S.E.Means followed by the same letter are not significantly different at p<0.05, as determined by Duncan"s test compared to uninoculated plants (Figure 1).In this case, study reported by Estaun et al. (2003), proved that different cultivars show different responses to the same fungus.Eftekhari et al. (2012) also found differences in the percentage of root colonization among four grape cultivars.Similarly, data recorded in this study reported a cultivar dependent impact of G. intraradices on olive cultivars that induced changes in the roots.
In other cases, Augé (2001) and Staddon et al. (2003) reported that symbiotic efficiency between the two partners is indirectly influenced by environmental factors at the community, organism and cellular level.In addition, Smith et al. (2004) claimed that the nutrient uptake and transport capacity of the symbionts can influence the symbiotic efficiency.The present study evaluated microscopic observations but not physiological and agronomic parameters, and this may be explained by the differences detected in both M% and A% between the five cultivars.
Differences in values of M% and A% were detected in the five Tunisian olive cultivars and to our knowledge the increase in each of the two parameters is of special interest and may provide important information.The present research proved also that the local cultivars of olive (O.europaea L.), Oueslati, is the most compatible cultivar with "G.intraradices" AM fungi compared to the other cultivars.This finding may be related to the genotypes of these cultivars and/or other factors when symbiosis is established.As usual, when discussing functional diversity, the gene expression is often touched.The recognition and the initiation of the symbiotic dialogue of the AMF and the host plant could be described as compatibility and it is genetically determined.In this case, the genetic diversity and composition of the extra radical mycelium is discussed as an important factor in the recognition process (Koch et al., 2006).Moreover, fungal hyphae secrete a signal to the roots that leads to a specific induction of the symbiosis program in roots that are in contact with the fungus, including the expression of symbiosis genes (Chabaud et al., 2002;Kosuta et al., 2003).Subsequently, only specific cells adapt to the formation of an aspersorium and the penetration conditions that are established (Genre et al., 2005).
As mentioned by Wang and Qiu (2006), there is a tendency for a given plant species to favor a specific colonization morphology.Many researches demonstrate the high variability in the symbiotic species and the different combinations of host plant and AMF (Burleigh et al.,2002;Lerat et al., 2003;Munkvold et al., 2004;Smith et al., 2004;Avio et al., 2006;Jansa et al., 2007).In these studies, there is a high functional diversity in AM symbioses when different combinations of host plant and AMF are established, and then they have different impacts on the morphology, efficiency and gene expression patterns of the symbiosis.In this case, AM are an heterogeneous associations, for each combination a specific plant and fungus species are connected with The current studies focused only on a single aspect, such as colonizing ability, compatibility, or symbiotic efficiency.Rouphael et al., (2015) reported that, scientists, industries and horticulturists need to collaborate to integrate AMF agricultural practice as an effective and sustainable tool for improving yield and product quality of horticultural crops.Future studies should focus on understanding the AMF species and environments interaction, in order to finally select the best combinations.

Conclusion
Microscopic observations showed best compatibility between AMF colonization and olive roots of Oueslati cv, compared with Meski, Jarboui, Chemlali and Chetoui cultivars.This study should be complemented with other studies thus to select cultivars best adapted to climate conditions.

Figure 2 .
Figure 2. The intensity of mycorrhizal infection (M%) in olive (O.europaea L.) tree roots of five Tunisian local cultivars (cvs Oueslati, Meski, Jarboui, Chemlali and Chetoui) (M) compared with non mycorrhizal plants (NM).Each value is the mean of three replicates ± S.E.Means followed by the same letter are not significantly different at p<0.05, as determined by Duncan"s test.