Diversity of Arbuscular Mycorrhizal Fungi of Durum Wheat ( Triticum durum Desf . ) Fields of the East of Algeria

In Algeria, in the semi-arid and arid areas cereal production is limited because of many constraints like lack of mineral elements (phosphorus and nitrogen), low organic matter and an insufficient water reserve due to the low rainfall. Most researches on durum wheat (Triticum durum Desf.) are undertaken to increase its productivity. In recent years several methods have been used including using microorganisms. Arbuscular Mycorrhizal fungi (AMF) appear to have important roles in plants in various soil conditions. In Algeria and particularly in the eastern regions, the diversity of AMF spores in the soil is so far little studied. In this context this study focuses on the study of the diversity and the abundance of AMF spores in Triticum durum Desf. fields in arid, semi-arid and coastal regions. Soil samples were collected and characterized and the spores isolated by the wet sieving methods were observed, counted and identified. The results of the physic-chemical characterization of all soil samples reveal a clay loam, loam clay and loam sandy soils with an alkaline pH (7.54-8.27), a total nitrogen content from 0.023 to 0.082% and phosphorus content ranging from 1.63% to 7.74%. We noted the presence of several morphotypes that were classified as follow: Glomeraceae, Acaulosporaceae and Scutellosporaceae with a predominance of Glomeraceae in different sites depending on climate and soil type. The presence of these families demonstrates a biodiversity of the eastern wheat fields of Algeria.


Introduction
Cereals are one of the most important crops in the world.They are a major source of food for both human's consumption and animal feed.In Algeria, the cereal and cereal derived food products occupy strategic place in our food system and our national economy as this culture covers more than 90% of the cultivated grounds (Djermoun, 2009).
The major cereal crop in our country is wheat which its production is exposed to various edaphic constraints (chemical, physical, biologic or climatic); especially those encountered in semi arid and arid regions.In research, several approaches are used for improving wheat production including AMF which are the principal symbiotic components of the soil edaphon and account for 25% of the biomass of the soil micro-fauna (Hamel, 1996).Indeed, wheat is easily mycorrhized by AMF under controlled greenhouse conditions and under field conditions (Cade-Menun et al., 1991;Neveen & Bahaa, 2011).These fungi have many advantages on the ecological level as well as the economic level, they establish symbiotic associations with plant roots, thus allowing the improvement of the agricultural products yield, and they translocate water and mineral nutrients such as phosphorus and nitrogen to the plants (Hu et al., 2010;Neveen & Bahaa, 2011).They positively affect plants tolerance to both abiotic (soil salinity and drought) as well as biotic (pathogens) stresses (Allen & Boosalis, 1983;Strullu et al., 1991).

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Isolation and Identification of AM Spores
The AM spores were isolated from 100 g of each soil sample by wet sieving and decanting technique of Gerdemann and Nicolson (1963).
The spore suspension has undergone a first centrifugation at 2000 RPM for 3 minutes in order to separate the spores from soil particles and root fragments, than a second centrifugation on a sucrose gradient at 2000RPM for 1 minute in order to collect spores of a similar size and density fraction.This spore suspension is poured into a Petri dish and each type of spores is sorted and counted under a binocular magnifying glass (Olympus SZX16).
All healthy spores were examined for their various morphological characters (color, form and size).The diameters of AMF spores are determined with an Image Pro Plus imaging system (4.0 version) attached to a compound microscope (Olympus BH-2).The AMF spores were identified with the help of identification manual of the of the Vesicular Arbuscular Mycorrhizal Fungi, certain species were also characterized on the basis of bibliographical data published on the web sites of INVAM (http://invam.caf.wvu.edu/fungi/taxnomy/speciesID) and BEG (http://www.kent.ac.uk/bio/beg/).

Control of Mycorrhization
Mycorrhization check was performed to confirm the level of colonization of wheat roots by AMF.Triticum durum Desf.Boussellem variety was provided by the Institut Technique des Grandes Cultures Khroub/ Constantine, Algeria (ITGC).The seeds were inoculated with the spores isolated from durum wheat fields (5 morphotypes) and put in plastic pots in the greenhouse at a temperature of 25±10 °C and relative humidity of 45±15% for 3 months.The effect of mycorrhization is determined by the staining method described by (Vierheilig et al., 1998), the entire root system of each plant was thoroughly rinsed with water and cleared in 10% KOH for 45 min at 90 °C and stained for 15 minutes in the ink solution.
After staining, 90 fragments of about 1 cm length were randomly selected and mounted between slide and cover, with 10 pieces per slide and examined under light microscopy.Mycorrhizal structures were observed (magnification 100) and for each root system showing at least one point of infection (penetration of hyphae in the root) is considered as mycorrhized.This technique calculates 5 parameters of infection by the method of (Giovannetti & Mosse, 1980).
We also calculated the frequency of mycorrhization calculated by MYCOCALC software.

Statistical Analysis
The results obtained from the physico-chemical analysis of all soil samples were treated statistically by the one-factor analysis of variance (ANOVA) and the comparisons of the averages of the parameters measured were performed at the threshold of 5 % by the test of Newman keuls with XLSTAT 2014 software.

Physico-Chemical Properties of the Soil
From the values of particle size of clay, silt and sand, we reported the soil texture of cereal fields of the eastern parts of Algeria.The results show a clay loam soil in the regions of Sétif, Tébess, Jijel and Annaba, a loam clay soil in the regions of Constantine, Oum El Baouaghi, Batna and Biskra, and finally a loam sandy soil in Khenchela.
The results presented in Table 2 show a near to 8 pH for all the soil samples, a 45% to 64 % rate of saturation, an electrical conductivity between (0.48 to 2.59 mS/cm -1 ) and total nitrogen content of 0.023 to 0.082 %.However, the phosphorus values are between 1.63 % and 7.74 %.After variance analyzes, significant differences between the different sites (P < 0.05) for all the parameters except the total nitrogen were revealed.Table 3 shows the number of extracted spores of 100 g of soil collected from the different sites surveyed in the eastern part of Algeria: 3-4 spores/100g of soil in the coastal zone, 5-12 spores/100g of soil in the semi arid zone and 6 spores/100g of soil in the arid zone.According to the analysis of variance of these results, no significant difference in the number of AMF spores were observed between arid, semi arid and coastal zones except for Khenchela (12 spores /100g of soil), Batna (11 spores/100g of soil) and Guelma (8 spores/100g of soil). jas.ccsenet.

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The In all the studied regions, the diversity of AMF was of a maximum 5 species found in the Batna region, which is characterized by a clay loam soil, 4 species of AMF were recorded in the region of Biskra characterized by a dry land.Glomus spores are among the most frequently found species.According to several authors, species of Glomus are distributed in different parts of the world and dominate the communities in arid, semi-arid and temperate zones (Li & Zhao, 2005;Gay et al., 2006;Uhlmann et al., 2006).And according to Pande and Trafdar (2004), the AMF dominate the alkaline and neutral soils under various edaphic conditions.
The observation of inoculated wheat roots by the isolated spores in pots and under greenhouse conditionsshow colonization by AMF.These fungi form characteristic structures such as arbuscules, different shapes of vesicles, intra and intercellular hyphae and spores.Furthermore, the vesicles presence inside cortical tissues (Figure 3) partly confirms the predominance of Glomus as they are the only kind that producesvesicles inside the host tissue (Smith & Read, 2008).
A high intensity of the mycorrhizal colonization of the roots of durum wheat inoculated by the 1, 3 and 4 (Glomus, Acaulospora) morphotypes was observed.In addition, the arbuscule abundance (a) was higher in roots inoculated with the morphotype 1 (Glomus) with a 60% rate but a very low arbuscule abundance of the mycorrized fragments was noticed (13%).This might be due to the short life of arbuscules (1 to 3 weeks) which degenerate and will be replaced by hyphas and vesicles that persist until the death of the cell.Our results are similar to those of Saad ( 2009).Thus the level of colonization of the roots by the 1 and 3 (Glomus) morphotypes are most significant and would be interesting for the production of inoculums.

Conclusion
Mycorrhizal associations play important roles in the functioning and the stability of different ecosystems.Many researchers have been studying the role of AMF in productivity.These studies have shown the importance, the diversity and abundance of mycorrhizal propagules.
This study focused on the diversity of AMF and characterization of the isolated spores from the surveyed areas.The characterization of the spores revealed the predominance of Glomus (Glomeraceae) and the presence of the two genera Acaulospora and Scutellospora.our work revealed the phenotypic description of the main spores found in the soil.
It would be interesting to continue this research highlighting the diversity of Glomales in the east of Algeria extracting significant numbers of spores for systematic studies in order to reinforce the role of mycorrhizal symbiosis as a biological agent improving the growth of cereals in the arid and semi arid areas, to produce adequate inoculums and to produce a bank of AMF.

Table 1 .
Principal localizations of the cereal sitesof the east area of Algeria

Table 2 .
Physico-chemical characteristics of the 10 soil samples