Distribution of the Arbuscular Mycorrhizal Fungi in AlJabal Alakhdar Area, East Libya

In order to investigate occurrence and distribution of Arbscular Mycorrhizal Fungi (AMF) in Aljabal Alakhthar area, east Libya. Roots and rhizosphere soil of 49 cultivated plants belonging to 7 families from 8 locations were collected. The percentage of root colonized by AMF was estimated. Spores extracted from soil samples were counted and morphologically identified to genus. The results indicate that all examined plants were colonized by AMF. Colonization rate and spore abundance differed according to location and plant. Colonization rate range from 97% in Petro selinum at Alqubbah location to 29 % in Vicia faba at Almarj. While spore abundance range from 992 spores / 100gm soil accompanied with Vicia faba at West alawilia location to 121 spores / 100gm accompanied with Avena sativa at Alqubbbah. As average of all locations, the plants belonging to Apiaceae had the highest colonization percentage where the plants belonging to Cucurbitaceae had the highest spore abundance. Lumloda location records the highest colonization rate. There was no correlation detected between root colonization rate and spore abundance. However, soil available phosphorus and clay percentage negatively correlated with degree of AM root colonization. Positive correlation was found between silt percentage and spore abundance. Glomus was the most abundant genus in all studied locations followed by Acaulosora, Gigaspora and Scutellospora


. INTODUCTION
Al Jabal Al Akhdar (Green Mountain) is located in approximately between latitudes 32° and 33° North and 20° and 23° East in the northeastern region of Libya and south Mediterranean Sea. It is 881m above sea level. It extends on the coast belt to about 300 km. The area receives total annual rainfall 250-600mm [1,2]. Although Al Jabal Al Akhdar covering only 1% of Libyan area. It consider the biggest and most Important Plant Area (IPA) in Libya as it contains 75 -80% of the Libyan flora and considered the most important area for agriculture in Libya [3]. 35% of the Al Jabal Al Akhdar area converted to crop areas mainly for grain , forage and vegetable production like wheat, barley and oats and 65% still the actual area of natural forest [4], [5].
Arbuscular mycorrhizal (AM) symbioses are formed between plant roots and fungi belonging of Glomeromycota [6]. Arbuscular Mycorrhizal Fungi (AMF) interactions predominate, which are found in about 80-90% of terrestrial plant families in natural and agricultural ecosystems [7].
AMF can colonize 300000 plant species belonging to 1000 genera followed 200 plant families [8]. Arbuscular mycorrhizal fungi are considered as obligate symbiotic biotopes, as they cannot grow without a host plant supplying them with carbohydrates [9], [10], [11]. In this symbiotic association, the fungus occurs inside cortex cell and also extend out in to surrounding soil as a hyphal network [9], [12]. In AM symbiosis, both organisms benefit as the fungus received photosynthesis products from host plant.The host plant receives a variety of benefits have already reported in earlier studies including: 1-Enhanced nutrient uptake over non -mycorrhizal controls [13] 2-improved water relations ( increase drought resistance) [14] 3-Increase pest and disease resistance [15], [16]. [17].

5-Increase
Efficiency of phytoremediation of contaminated water and soil [18].
6-Increase efficiency of nitrogen fixation by Rhizobium [19] 7-Enhancement of soil aggregation and stability [20] Because of the public concerns about the side effects of pesticide and chemical fertilizer, major attention has been given to research areas concerning arbuscular mycorriza as biofertilizer and biocontrol agent.Despite the importance of arbuscular mycorrhiza in agriculture and forestry, there is no work has been done regarding their presence, distribution and diversity in the Aljabal Alakhdar area. The objective of this study is to evaluate the occurrence and distribution of FAM accompanying agricultural crops in AL Jabal Al Akhthar area.

Root and soil samples collection
Roots and rhizosphere soil samples were collected from the most common 49 cultivated plant (3 replicate), from 8 locations in Al-Jabal Al-Akhdar. These sampling locations were chosen due to their high agricultural density Fig. (1), Table (1). The roots were fixed in formalin-acetic acidalcohol (FAA; 10:35:10:5Formalin-water-ethanol-acetic acid) as soon as possible and were kept with soil samples at 5°C for further analysis

Soil Analysis
Soil chemical and textural characteristics were determined as following. Soil texture by hydrometer method, Soil Reaction (pH) by pH meter, CEC by the sodium acetate-method. The total nitrogen by kjeldahl method, Organic Content by rapid titration method [21], available phosphorus by Olsen's method [22], Ca and Mg by titration with EDTA (23), Potassium and sodium by flame photometer [24]. The main characteristics of the soil are recorded in Table 2.

Assessment of AMF colonization
Root fixed in FAA and washed with water 3 times. The percentage of mycorrhizal colonization of roots were estimated by cutting the roots into 1cm pieces and clarified with 10%(w/v) KOH at 90ºC in water bath for 20-30 minutes and acidification by HCl (5%) for one minute and staining with trypan blue at 90ºC for ten minutes [25]. The stained roots placed on the glass slides for microscopic observations under 200×magnifications . AMF colonization was estimated by examination one hundred pieces of roots for each sample. Percent root colonization was counted according

Recovery and counts of AM fungal spores
AMF spores were extracted from 100 g Rhizosphere soil by wet sieving and decanting [26] followed by sucrose gradient centrifugation and the Spore density (number of spores /100g soil) was counted. The spores were distinguished into morphotypes and identification up to genus level based on spore size, color, shape, hyphal attachment and spore ornamentation under a stereomicroscope using the descriptions provided by [27] , [28]. The Relative abundance (RA) was counted according Total number of spores observed

Statistical analysis
ANOVA and correlation analyses were carried out. The means were compared using Least Significant Difference (LSD) at p <0.05 after, ANOVA. The relationships between AMF parameters and soil properties were determined using Pearson's correlation analysis.    Fig. (3). There was no correlation between root colonization degree and spore abundance and these results are consistent with several studies [30], [31], [32]. Also, no correlation was found between soil available phosphorus and spore abundance. However, negative correlation was found between soil available phosphorus and degree of root colonization (r= -0.6186). In earlier studies [33], [34], [35] obtained the same results. This negative correlation could be due to changes in permeability of root cell membrane. In earlier study [36] the researchers have concluded that the phospholipid levels in root cell wall were correlated with the percentage p content of root tissue and amount of p added to soil. Root exudation, soluble amino acids and reducing sugars from the roots depend upon the phospholipid level in roots and associated changes in permeability properties of root membranes. When the phosphorus increase in soil and plant tissue, lead to increase of phospholipids in root cell membrane and decrease of permeability and root exudation and decrease colonization rate. Also, negative correlation was found between clay percentage and colonization degree (r= -0.71) while positive correlation was found between silt percentage and number of spores and (r=0.74). As average of all locations, Fig.(4) shows that the plants belonging to Apiaceae had the highest colonization percentage with 93.5% followed by Labiatae 86%, Fabaceae 73%, Asteraceae 72%, Amayllidceae 71.83%, Poaceae 71.53%, Cucurbitaceae 67.75% and Solanaceae 63.5%. Fig.(5) also shows that the plants belonging to Poaceae had the highest colonization rate in Albalenge, Alwasita, Almarj, and Alawilia with 79.83%, 78.75%, 77% and 71% respectively. However, the plants belonging to Fabaceae had the highest colonization rate in West awailia and Alhaboon locations with 92% and 75.5% respectively. In Lumluda and Alqubbah locations, the plants belonging to Solanaceae and Apiaceae had the highest colonization rate with 96% and 93.5% respectively.  Our results show that there was variation in AMF colonization and spore abundance between studied locations and plant families which could be due to one or more of multiple factors that have been reported already in several studies including:
3. AMF factors. The differential sporulation ability of AM and the dormancy [44] 4. Local ecological factors such as plant cover and host diversity, soil cultivation and disturbance and seasonality [45], age of the host plants, host dependence of AMF species [46], hostspecificity between fungi and plants [47].
Glomus genus had the highest spore proportion of 53.13% followed by Acaulosora with 26.87%, Gigaspora 11% and Scutellospora 9%. Glomus was dominate in all locations and the relative abundance of Glomus range from 62% at west awailia to 42% at Alhabbon location, where the relative abundance of Acaulospora range from 34% at Almarij to 24% at Alwasita . The relative abundance of genera Gigaspora and Scutellospora range from 19% at Albalenge and 16% at Algubbah respectively to 12% at Almarij and 5% at Albalenge respectively.  Glomus was the most abundant genus and had the highest spore proportion in all studied locations and plant families, similar results were observed in other places by [31], [46], [51].
Several studies have suggested that Glomus present anywhere due to the high adaptability of Glomus to different soil conditions, host and environment. [51], [52], [53].