Spatiotemporal variation of arbuscular mycorrhizal fungal colonization in olive (Olea europaea L.) roots across a broad mesic-xeric climatic gradient in North Africa

Science of the Total Environment 583 (2017) 176–189

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Spatiotemporal variation of arbuscular mycorrhizal fungal colonization in olive (Olea europaea L.) roots across a broad mesic-xeric climatic gradient in North Africa Amel Meddad-Hamza a, Nabila Hamza a, Souad Neffar b, Arifa Beddiar a, Silvio Gianinazzi c, Haroun Chenchouni b,⁎ a b c

Laboratoire de Biologie Végétale et Environnement, Department of Biology, Faculty of Sciences, University of Badji Mokhtar, Annaba 23000, Algeria Department of Natural and Life Sciences, Faculty of Exact Sciences and Natural and Life Sciences, University of Tebessa, Tebessa 12000, Algeria INOCULUMplus Technopôle Agro-Environnement RD31, Bretenière 21110, France

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Olive root colonization by arbuscular mycorrhizal (AM) fungi was studied across a broad climatic gradient in Algeria. • Mycorrhizal intensity and spore density increased with the increase of precipitation and the decrease of temperature. • The variety Rougette had the highest seasonal mycorrhizal parameters in all plantation ages and climates. • Spore community included six genera Rhizophagus, Funneliformis, Glomus, Septoglomus, Gigaspora, Scutellospora and Entrophospora. • MPN was positively correlated with spore density in rhizosphere soil of olive grown under different North African climate zones.

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Article history: Received 30 September 2016 Received in revised form 8 January 2017 Accepted 8 January 2017 Available online 13 January 2017 Editor: P Elena PAOLETTI Keywords: Arbuscular mycorrhizal fungi diversity North-African climates Olive varieties Plantation age Seasonal dynamics

a b s t r a c t This study aims to determine the spatiotemporal dynamics of root colonization and spore density of arbuscular mycorrhizal fungi (AMF) in the rhizosphere of olive trees (Olea europaea) with different plantation ages and under different climatic areas in Algeria. Soil and root samples were seasonally collected from three olive plantations of different ages. Other samples were carried out in productive olive orchards cultivated under a climatic gradient (desertic, semi-arid, subhumid, and humid). The olive varieties analysed in this study were Blanquette, Rougette, Chemlel and the wild-olive. Spore density, mycorrhization intensity (M%), spore diversity and the most probable number (MPN) were determined. Both the intensity of mycorrhizal colonization and spore density increased with the increase of seasonal precipitation and decreased with the increase of air temperature regardless of the climatic region or olive variety. The variety Rougette had the highest mycorrhizal levels in all plantation ages and climates. Spore community was composed of the genera Rhizophagus, Funneliformis, Glomus, Septoglomus, Gigaspora, Scutellospora and Entrophospora. The genus Glomus, with four species, predominated in all climate regions. Spores of Gigaspora sp. and Scutellospora sp. were the most abundant in desertic plantations. Statistical models indicated a positive relationship between spore density and M% during spring and winter in

⁎ Corresponding author. E-mail address: [email protected] (H. Chenchouni).

http://dx.doi.org/10.1016/j.scitotenv.2017.01.049 0048-9697/© 2017 Elsevier B.V. All rights reserved.

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young seedlings and old plantations. A significant positive relationship was found between MPN and spore density under different climates. For a mycotrophic species, the rhizosphere of olive trees proved to be poor in mycorrhiza in terms of mycorrhizal colonization and numbers of the infective AMF propagules. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Fungi are one of the main components of the soil microbial community, as they are dominant in aerobic environments (Alguacil et al., 2011; Montes-Borrego et al., 2014) with a biomass (that of saprophytes and mycorrhizae) ranging between 50 and 900 kg C/ha (Zhu and Miller, 2003). They play a crucial role in soil biology and therefore the functioning of ecosystems, through numerous features such as (i) decomposition of organic matter and the release of nutrients available for absorption by plants (Zhu and Miller, 2003; Wilson et al., 2009), (ii) storage of nutrients within bodies of soil organisms preventing loss of nutrients through leaching, and (iii) maintenance of soil structure and regulation of gas balances and biogeochemical cycling of soil (Wang et al., 2007). The composition and activity of the soil microbial community affect ecosystem processes (Hawkes et al., 2005), as well as their stability and fertility (Smith and Papendick, 1993), which can be a sensitive indicator of changes in soil quality, especially in Mediterranean regions where climatic and lithological conditions with landscape structures and human activities are responsible for the increasing desertification and land degradation (Chenchouni et al., 2010; Hedo de Santiago et al., 2015). That said, soil biology has proven to be a core link and a key-challenge in the assessment of soil resilience aptitude especially with regard to current climate change (Allison and Martiny, 2008). Given these many vital functions, soil management practices improve soil fertility, water and nutrients retention, edaphic biodiversity, and production of durable crops (Cerdà and Jurgensen, 2011; Neffar et al., 2013). According to Vasconcellos et al. (2013), biological indicators of soil quality are the first attributes that undergo changes following the degradation of the habitat. The arbuscular mycorrhizal (AM) fungi are major components of the edaphic rhizosphere where they are capable of establishing symbiotic associations with the fine roots of almost 80% of terrestrial plants (Dandan and Zhiwei, 2007). In this symbiosis, the AM host plant provides carbon sources and the fungus increases: (i) the absorption capacity of some elements such as phosphorus and nitrogen (Ji et al., 2010), (ii) plant resistance against pathogens (Linderman, 2000), (iii) tolerance to drought by affecting water balance, where both avoidance and tolerance to dehydration are involved especially in arid and semi-arid ecosystems (Augé, 2001), (iv) tolerance to soil pollution and salinity (Porras-Soriano et al., 2009), (v) the fungus accelerates the recovery of vegetation in disturbed arid ecosystems (Caravaca et al., 2003) and (vi) AM fungi, through the hyphae and glomalin produced, alter the vital attributes of soil, forming stable macroparticles, and increase resistance to erosion (Wilson et al., 2009) and stimulate microbial growth in soil (Bai et al., 2009). They play a major role as biofertilizers within vegetation, as well as bioprotectants and bioregulators (Gianinazzi and Wipf, 2010). Currently, studies focus on the role played by the high diversity of these fungi in soil systems and their uses in improving crop productivity, soil fertility and the ecosystem sustainability (Caravaca et al., 2005; Alguacil et al., 2011; Mengual et al., 2014). Actually, diversity and community structure of AM fungi are important to maintain the stability and recovery of plant communities in disturbed habitats of arid and semiarid lands (Dandan and Zhiwei, 2007). As these ubiquitous and generalist fungi are associated with the majority of plant families in different ecosystems around the world (Gai et al., 2006), some AM fungal species show a preference or a narrow specificity regarding host symbionts (Bidartondo et al., 2002). In addition, for the same taxa, mycorrhizal presence varies depending on the AM fungal community composition,

geographical location (Ranelli et al., 2015; Montes-Borrego et al., 2014) and abiotic conditions of soil (Xiang et al., 2014; Neffar et al., 2015). Fluctuations in environmental factors such as, temperature and soil moisture affect the diversity, coexistence, and structure of soil microbial populations and communities (Torrecillas et al., 2013; Montes-Borrego et al., 2014). In addition, an increase in soil moisture as a result of rainfall leads to increased soil hyphal abundance with associated change in soil structure following increases in soil aggregation, humification, and organic carbon (Wilson et al., 2009). Rainfall or soil moisture have been found to positively influence fungal abundance in agricultural and woody lands (Frey et al., 1999; Querejeta et al., 2009). In the case of AM fungi, spore abundance and colonization are significantly affected by abiotic factors. For example, sandy soils reduce fungal spores and thus limit the distribution of fungal communities in dunes, because root colonization is affected by moisture (Hatimi and Tahrouch, 2007). On the other hand, Bohrer et al. (2004), minimize the influence of abiotic factors on mycorrhizal parameters and attributed seasonal variations to the host species phenology. Olive (Olea europaea L.) is a mycotrophic species adapted to drought and poor soils (Meddad-Hamza et al., 2010; Mekahlia et al., 2013). In Algeria, the species is cultivated along a large climatic gradient stretching from humid coastal areas through the semi-arid high plateaus to the hot hyperarid climate in the Sahara Desert in the south. Indigenous plant to Mediterranean region, the wild-olive (Olea oleaster) is found as dominant species or mixed with other species over large woody and shrubby mountain habitats. The common varieties of olive grown in Algeria are: Blanquette and Rougette in Guelma in northeastern Algeria, Sigoise in Oran in northwestern Algeria (Sidhoum and Fortas, 2013), and variety of Chemlel in the Soummam Valley (Khelfane-Goucem, 2001). In Algeria, olive plantations occupy 200,000 ha, i.e. 45% of the national arboricultural area, and produced about 578,740 tons of oil in 2013 (FAOStat, 2015). Despite the major role of mycorrhizae for olive, explorations of AM diversity in various olive agroecosystems and the applications of AM are recent. In the Mediterranean region, Spanish investigations are the most numerous; these studies focused on AM fungal biodiversity, the application of mycorrhizae for the development of two olive varieties (Arbequina and Leccino), and the relationships between AM fungi and abiotic stresses (Calvente et al., 2004; Porras-Soriano et al., 2006; Porras-Soriano et al., 2009). In North Africa, olive mycorrhizal studies are rare. These investigated mainly (i) effects of AM fungal biodiversity on growth of olive trees (Kachkouch et al., 2012; Chliyeh et al., 2014), (ii) the role AM fungi in alleviating oxidative stress induced by drought on the Picholine variety (Fouad et al., 2014), (iii) the mycorrhizal status of the wild olive (Olea oleaster) (Sghir et al., 2013), and (vi) the effect of irrigation by wastewater from Tunisian olive mills on the functioning of olive AM fungi (Mechri et al., 2011). In Algeria, the relationship olive– mycorrhiza remains little understood. The noteworthy published works, conducted only in northern Algeria, tested the effect of AM inoculation on the growth and resistance to transplant stress of olive seedlings (Meddad-Hamza et al., 2010); the effect of seasons across a climatic gradient on the variation of AM colonization in olive roots of the variety Ferkani (Mekahlia et al., 2013); and the improvement of tree growth of olive orchards in western Algeria by two isolated Glomus species (Sidhoum and Fortas, 2013). Because very little information is available on the dynamics of AM fungi associated with olives grown under different climatic regions in North Africa, this study aims to determine how the spatiotemporal dynamics of mycorrhizal parameters (mycorrhization intensity and

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density of spores) vary following plantation age and olive varieties in northeastern Algeria. Thus this approach establishes the mycorrhizal status of each variety and reveals olive growth stages, based on plantation age, that require higher mycorrhization to maintain standard and balanced physiological performances. Moreover, the survey aims to elucidate the effects of climatic variables on mycorrhizal intensity, spore density, diversity of spore spectrum and mycorrhizal potential (Most Probable Number) of olive rhizosphere across a climatic gradient that includes desertic, semi-arid, subhumid and humid climates. 2. Material and methods 1. Study sites and stations The study sites of olive plantations are located along a climatic gradient north–south of Algeria (Fig. 1). Four sites were selected to explore mycorrhization (mycorrhizal intensity, density and diversity of AM fungal spores and soil mycorrhizal potential) according to bioclimatic regions. These sites are located under different climatic conditions ranging from humid climate in the North to desertic in the South (Fig. 1, Appendix 1). The olive orchards sampled within each site included two varieties of Olea europaea (Chemlel and Rougette) and wild-olive (Olea oleaster). With the exception of the orchard of El Oued, completely bare, the herbaceous layer based on annual species is dominant in the different study sites. The main species include Cynodon dactylon, Trifolium campestre, Galactites elegans and Erodium cicutarium. • Site of El Oued (33°21′11″ N, 6°47′44″ E, altitude = 60 m a.s.l.) is characterized by a desertic climate with two seasons, a mild-dry winter and a hot-dry summer. The average temperatures reach 40 °C in summer and range from 2 to 20 °C during winter. Rainfall in the area is irregular and very scarce, often not exceeding 100 mm/year. The 2-ha olive orchard studied was of 50% Rougette, 45% Chemlel and 5% Olea oleaster. The soil of the study orchard is characterized with sandy texture and very poor organic matter. The understory is basically absent (Appendix 1). • Site of Tebessa (35°28′02″ N, 7°58′09″ E, altitude = 880 m a.s.l.) is located in the semi-arid region and characterized by warm dry summer

and cold winter. Average temperatures range from 11 to 35 °C during the hot season and from 2 to 15 °C in winter. On average, 400 mm precipitation is recorded annularly. The orchard of olive examined (1 ha) composed of 50% variety Chemlel and 50% variety Rougette, with a plot of some wild olive trees. Soils have a silty clay texture with 4.1% of organic matter. The orchard understory was occupied by herbaceous spontaneous vegetation (Appendix 1). • Site of Guelma (36°37′27″ N, 7°40′35″ E, altitude = 160 m a.s.l.) located under subhumid climate with cold-rainy winter (rainfall = 450– 650 mm/year). Mean temperatures average 15–30 °C in summer and 6–15 °C in winter. The rainy period lasts 6–7 months a year with an average rainfall of 614 mm/year. – For the climatic gradient study, the sampled olive plantation covered an area of 2.5 ha with a planting density of 100 trees/ha. The varieties Chemlel and Rougette occupy respectively 30% and 70% of the plantation. Near the study orchard lies twenty wild-olive trees. Soils were characterized by silty and silty clay textures with a pH of 8.05 and an average organic matter of 3.26% (Appendix 1). – For the study of the effects of plantation age, seasons and varieties, three different stations, planted with two varieties of olive Blanquette and Rougette were sampled: (i) an old plantation (N 100 years) located in the municipality of Bekouche Lakhdar (36°42′23″ N, 7°17′ 49″ E) in the wilaya (province) of Skikda, (ii) a young plantation (6–7 years) located in Ain Ben Beida (36°37′27″ N, 7°40′35″ E) near Bouchegouf (Guelma), and (iii) young olive seedlings (~1 year) of the plant nursery located in Belkhir (36°27′58″N, 7°29′55″ E) in Guelma (Fig. 1). Samples collected from these three stations were used to determine seasonal variations of AM colonization of roots and AM fungal spore density of the soil. • Site of Bejaïa (36°34′53″N, 5°40′05″ E, altitude = 740 m a.s.l.) is located under humid climate. Rainfall varies between 670 and 1300 mm/year, and spread mostly over September–March. Mean temperatures recorded 16–29 °C in summer and 5–20 °C in winter. The sampled plantation is located at the experimental station of the “Technical Institute of Arboriculture”, where soils have a silty clay texture with a pH = 8.04 and organic matter = 1.39%. The station stretches over 1.5 ha in which the varieties Chemlel and Rougette are planted in lines, with 50% each. The wild-olive is planted on the edge of the orchard with 30 trees. The

Fig. 1. Geographic location of sampled sites (●) across a climatic gradient in northeastern Algeria. Dashed lines delineate climate patterns: hot hyperarid or desertic (El Oued), semi-arid (Tebessa), subhumid (Guelma, including three stations, BK: the plant nursery in Belkhir, BB: Ain Ben Beida, and BL: Bekkouche Lakhdar), and humid (Bejaia). Lateral diagrams represent climate data (mean temperatures in solid squares and precipitation in bar chart) of each study site. Solid triangles (▲) indicate locations of the closest meteorological station to each study site.

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understory of the study orchard is dominated by herbaceous plants (Appendix 1).

2. Soil and root sampling In order to study the seasonal variation of mycorrhization parameters within both varieties Blanquette and Rougette at Guelma, soil samples (~500 g) including roots of ten olive trees were randomly collected between the upper soil horizon and 25 cm depth in each of the three study stations: Bekkouche Lakhdar (BL), Ain Ben Beida (BB) and the plant nursery Belkhir (BK). In the four seasons of 2006, six soil samples were taken under each tree of each variety to estimate spore density, whereas these samples were pooled per tree to determine AM intensity. As for the change in mycorrhizal parameters (diversity and density of fungal spores, and most probable number “MPN”) among different climates, soil samples (~500 g) with the fine roots were randomly collected in autumn 2006 under five adult trees of each variety (Chemlel and Rougette) and wild-olive. Soil samples were taken at three points around the trunk of each tree (within a 1 m radius) at a depth of 25 cm. Most fine rootlets were collected at the same time as the soil. The three samples were pooled to have a sample per tree (i.e. five sample per variety) that was used for the estimation of AM intensity. For each olive species and variety, the five samples of soil/roots were pooled to have a composite sample that was used to determine the density of fungal spores and the most probable number “MPN” of propagules. 3. Estimation of AM root colonization Mycorrhization was estimated using the method of Phillips and Hayman (1970). Root fragments were washed, treated with 10% KOH to empty radicular cells and then bleached with H2O2 and 20% HCl for 1 h. The bleached roots were then stained for 20 min with 0.03% Trypan blue solution at 90 °C. The level of colonization was appraised using the method of Trouvelot et al. (1986). From the soil sample of each season and each variety, thirty root fragments of about 1 cm length were randomly selected. The latter were arranged in parallel on glass slides at a rate of fifteen fragments per slide, in a drop of glycerol. Each sampled tree was represented by one microscopic slide (i.e. 10 slides per variety for each season in stations of Guelma, and 5 slides for each climatic region). Examination with light microscope (×40) allowed to annotate each fragment based on a scale of classes. This scale estimates the level of mycorrhizal colonization of each fragment by means of six classes and classifies arbuscular abundance into four classes. These observations were used to fill an evaluation grid to calculate the mycorrhizal colonization using software MycoCalc (http://www2.dijon. inra.fr/mychintec/Mycocalc-prg/download.html). 4. Identification and enumeration of AM spores in olive rhizosphere A comprehensive enumeration of fungal spores in 50 g of soil was also performed for each soil sample (6 samples per sampled tree × 10 trees = 60 samples per variety × 2 varieties = 120 samples per season × 4 seasons = 480 samples per station × 3 stations = 1440 samples at the site of Guelma). For the different climates, one composite sample was analysed per olive variety/species. Fungal spores were extracted using the wet sieving technique (Gerdemann and Nicholson, 1963). Spores were mounted on glass slides in a drop of biochemical reagents containing the Polyvinyl-Lacto-Glycerol (PVLG) and a mixture of Melzer's reagent and PVLG (1:1 V/V), and then observed using microscope for species identification following phenotypic characters (Blaszkowski, 2008). The identification of certain species was confirmed using molecular characterization at the Laboratory of Molecular Biology of plant-microbe relationships JESRF-INRA, Dijon, France. Twenty spores of each

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morphotype of AM spore were rinsed several times with distilled water then placed in 1.5 mL Eppendorf tubes. Spores were gently split with a small pot to extract the DNA which was purified using the NucleoSpin Plant kit (Macherey-Nagel, Germany) and then recovered in 50 μL of ultrapure water. Double amplification (Mullis et al., 1986) had amplified the ribosomal DNA (rDNA) of the spores using universal eukaryotic primers: ITS1 (White et al., 1990) and NDL22 (van Tuinen et al., 1998), then more specific primers for fungi: ITS3 (White et al., 1990) and FLR2 (Trouvelot et al., 1999). The product of the 2nd PCR was used for cloning using the cloning kit “Chemo-competent bacteria Escherichia coli Top10cells” (Invitrogen Inc., Paisley, UK). The purification of the plasmid was carried out using Plasmid DNA Purification NucleoSpin kit (MachereyNagel, Germany). The sequencing was carried out by MWG Biotech Inc., Germany (www.eurofinsgenomics.eu). The obtained sequences were compared with the sequences known in the databases of the National Center of Information of the Biotechnology (NCBI: www.ncbi.nlm.nih. gov) at the nucleotide level using the computer software Basic Local Alignment Search Tool (BLAST: www.ncbi.nlm.nih.gov/guide/sequenceanalysis/) to identify homologous sequences. These sequences were aligned with clearly identified taxa sequences using the CLUSTALW software (Gollotte et al., 2004). A phylogenetic tree was constructed using the Neighbor Joining (NJ) algorithm, and then the robustness of the tree was tested by the bootstrap method. 5. Similarity of fungal species diversity between climates Jaccard similarity index (CJ) was used to compare spores of AM fungal species between sites taken in pairs. Given two sites, A and B, CJ was computed as: CJ = c / (a + b − c) × 100. Where a and b = the total number of species present in sites A and B, respectively; c = the number of species found in both sites (Magurran, 2004). 6. Evaluation of the most probable number (MPN) of mycorrhizal propagules The number of mycorrhizal propagules was estimated using the technique of the most probable number (MPN), which was adapted for AM fungal propagules by Poter (1979) and is based on the use of a series of successive soil dilutions at the rate of 10 (1/10, 1/100, 1/ 1000, 1/10,000 and 1/100,000) to gradually deplete the soil and thus identify the limiting dilution in which the existing propagules are no longer able to establish mycorrhiza. The dilutions were prepared by mixing the original soil with the same soil autoclaved at 120 °C for an hour (Gianinazzi-Pearson et al., 1985). The soil mixture is divided into five replicates of 50 g per pot. Pregerminated clover seeds were planted with one seedling per pot, transferred under controlled conditions in a greenhouse (average daily temperature 18–22 °C, with 60–70% relative humidity). The seedlings were watered daily with distilled water. After six weeks, the entire root system was stained according to the aforementioned technique (cf. Section 3). Using mycorrhizal and nonmycorrhizal roots obtained for each level of dilutions and for the five repetitions, the number of propagules present in soil was evaluated with the help of the table of Cochran (1950). 7. Meteorological data In order to investigate effects of climate conditions on mycorrhizal intensity, daily mean temperatures (t) and daily precipitation data from the meteorological sites of Bejaia, Guelma, Tebessa and El Oued were used. Raw daily data for three months (90 days) prior to the date of mycorrhizal estimation were used to calculate the following seasonal parameters: – T: average of mean air temperatures (°C), – PP: cumulated of daily precipitation (mm), – PP/T: average of hygrometry calculated as the ratio between

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precipitation (PP) and mean temperatures (Τ) (Idder-Ighili et al., 2015), – K: hydrothermal coefficient of Sielianinov (K = 10 × PP / (∑ t)), where PP is the total precipitation of the season and t is the average daily temperature (Wilczewski et al., 2012), – IDM: aridity index of De Martonne (IDM = PP / (T + 10)) (De Martonne, 1925). 8. Statistical analysis The variation in mycorrhizal intensity and spore density within three stations of Guelma was tested using the analysis of variance (three-way ANOVA). These linear models tested the effects of three factors namely: age of plantations (three categories), seasonality (four seasons) and olive variety (Blanquette and Rougette). Collinearity was tested between explanatory climatic variables by means of a correlation matrix built with seasonal data. The multicollinearity was checked for the climatic variables (Fig. S1), which lead to the use of only the seasonal mean temperature and precipitation as predictable variables for modelling the seasonal variation of mycorrhizal intensity; because the rest of climate parameters ‘PP/T, K, IDM’ were positively correlated with precipitation and negatively correlated with temperature. After that, the effects of air temperature, precipitation, plantation age and olive variety of the variation of M% and spore density were tested using generalized linear models (GLMs). Interactions between the climatic parameters (temperature and precipitation) the two factors (plantation age and olive variety) were included in each GLM. Mycorrhizal intensity fitted to a Gaussian distribution error with identity link, whereas spore density fitted using Poisson family with log link. The effects of explanatory variables were summarized using type-III F-test for M% and likelihood ratio ‘LR’ (Chi-squared test) for spore density. For GLMs, observations were not independent with respect to climatic variables and inferences about tree ages and climate zones are made without sampling replicate plantations within them. Simple linear regression (SLR) were applied to evaluate the relationship between the density of fungal spores and mycorrhizal intensity (M%). Since both variables are subjected to both spatial and temporal variations (Dandan and Zhiwei, 2007), these linear models were tested using the values observed in each of the three stations of Guelma (different plantation ages) and during the four seasons. In addition, two-way ANOVA was used to test the significance of changes in mycorrhizal intensity among the four climatic regions and olive varieties (three categories). For the latter factor, wild-olive was included in the ANOVA as a term of the factor ‘variety’ with those of Olea europaea (Chemlel and Rougette). In addition, the effects of temperature, precipitation and olive varieties on the variation of M% along the climatic gradient was tested using GLM with Gaussian distribution error and identity link. In three-way and two-way ANOVAs, all possible interactions between factors were included in the model. Significant ANOVA were followed by multiple comparisons of means using Tukey HSD test. Furthermore, the variation of spore density in soils and the MPN between climatic regions was tested using one-way ANOVA. Finally, the relationship between spore density and MPN along the four climatic regions of northern Algeria was modelled using a GLM. Climate type was inputted as explanatory variable to test whether this relationship differed between climates. Models and statistical tests were computed in R and plots were drawn using the package {ggplot2} (Chang, 2013). 3. Results 1. Seasonality of AM fungal parameters following plantation age and varieties 1. Mycorrhizal intensity Over all plantation ages, mycorrhizal intensity in both olive varieties was 42.7 ± 21.6% (mean ± SD). Mycorrhizal levels were higher in the

spring and autumn compared to other seasons Regardless of the plantation age or olive variety. Young olive seedlings (BK) showed 74% of mycorrhization in autumn and 67% in spring for the variety Rougette, whereas the mycorrhization in variety Blanquette was 73% in autumn and 61% in spring. In young plantations of 6–7 years (BB), mycorrhizal intensity remained high for both varieties in autumn and spring with 56% in autumn and 64% in spring for Rougette, whereas variety Blanquette recorded a value of 51% in autumn and 60% in the spring. As for the old plantation (BL), a slight decrease in mycorrhization was observed during autumn with 45% for Rougette and 49% for the Blanquette while M% remains above 50% in spring. The lowest M% values were observed in winter for variety Rougette with 24%, and in summer with 5% for Blanquette (Fig. 2A). Analysis of variance revealed that levels of mycorrhizal intensity significantly varied among olive varieties (F(1, 217) = 17.09, P b 0.001), plantation ages (F(2, 217) = 136.85, P b 0.001), seasons (F(3, 217) = 442.118, P b 0.001) and the interactions ‘Age × Saison’ (F(6, 217) = 10.44, P b 0.001) and ‘Age × Season × Variety’ (F(6, 217) = 3.96, P b 0.001). The interactions ‘Season × Variety’ (F(3, 217) = 1.37, P = 0.254) and ‘Age × Variety’ (F(3, 217) = 2.21, P = 0.112) were not significant. Tukey's test indicated that average of mycorhisation in Rougette (M% = 47%) was significantly higher than that of Blanquette (M% = 43%). Similarly, M% was significantly higher in roots of young trees of BK (M% = 54%) compared to that observed in the BB plantations (M% = 45%) and BL plantations (M% = 35%) which had significantly the lowest mycorrhization. In addition, Tukey's test identified three homogeneous groups of seasons. The average of mycorrhizal intensity recorded in summer (M% = 17%) was significantly lower than that of winter (M% = 42%). While mycorrhization in spring (M% = 61%) and autumn (M% = 58%) were significantly higher compared to other seasons. 2. Spore density The highest value of spore density was observed in spring in young plantation (BB) for both variety Rougette with 1196 spores and Blanquette with 1240 spores/10 g of soil. The same seasonal pattern was observed with a high density in old plantations (BL) with 1170 spores for Rougette and 1093 spores for Blanquette. Similarly, the number of spores decreased in young seedlings (BK) with 810 spores for Blanquette and 940 spores for Rougette during the same season. Spore density decreased in descending order by summer, autumn and then winter where the lowest values were observed with 100 to 300 spores/10 g of soil on average for both varieties (Fig. 2B). ANOVA revealed significant effects for the variation of spore density following the factors: plantation age (F(2, 120) = 25.57, P b 0.001), season (F(3, 120) = 1566.63, P b 0.001) and the interactions ‘Season × Variety’ (F(3, 120) = 2.85, P = 0.040), ‘Age × Season’ (F(6, 120) = 54.21, P b 0.001) and ‘Age × Season × Variety ‘(F(6, 120) = 27.21, P b 0.001). However, the ANOVA indicated a non-significant variation among varieties (F(1, 120) = 1.57, P = 0.21) and the interaction ‘Age × Variety’ (F(2, 120) = 1.08, P = 0.34). Multiple comparisons of means of spore density revealed three distinct groups of plantation ages. The average of spores counted in young plantations ‘BB’ (648 spores/10 g of soil) was significantly the highest. While it significantly decreased in young seedlings ‘BK’ with 569 spores/10 g of soil. The average number of spores recorded in the old plantation ‘BL’ was classified as intermediate with 612 spores. For the factor season, Tukey's test distinguished four homogeneous groups. The mean number of spores determined in winter was significantly lowest (222 spores/10 g of soil), and that got in spring was the highest with 1075 spores. Values noted during autumn and summer were classified intermediary groups with respectively 473 and 668 spores/10 g of soil. 3. Effects of seasonal climatic variables The GLM revealed significant effects of the variables ‘air temperature’ and ‘precipitation’ combined with ‘plantation age’ and ‘variety’ on the variation of mycorrhizal intensity (P b 0.001). Besides, the effects

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Fig. 2. Effect of plantation age on seasonal variation of mycorrhizal intensity (A) and spore density (B) in the rhizosphere of olive varieties Blanquette and Rougette cultivated in northeastern Algeria. Bars represent mean values ± standard deviation. (Win: winter, Spr: spring, Sum: summer, Aut: autumn).

of different interactions of these four variables were significant (Table 1). In both study varieties, M% significantly increased with the increase of precipitation (P b 0.001), but significantly decreased with the increase of temperature (P b 0.001). However, the model indicated that M% did not vary significantly between varieties under the effect of temperature (P = 0.158) or precipitation (P = 0.800), i.e. M% in both varieties varied in the same direction under the effect of the two climatic parameters (Fig. 3; Table 1). Spore density averaged 601.1 ± 313.5 spores/10 g of soil over the study plantations. Regardless of the age of olive plantations, spore density increased with the increase of Table 1 Generalized linear models (GLMs) testing the effects of olive plantation ages (~1 year, 6–7 years, and N100 years), olive varieties (Chemlel and Rougette), seasonal temperature and precipitation on the variation of mycorrhizal intensity (M%) and spore density in the rhizosphere of olive orchards cultivated in the Basin of Guelma, northern Algeria. (Df: degrees of freedom, F: F-statistics, χ2: Chi-squared value, P: P-value). M%

Spore density

Variables

Df

F

P

χ2

P

Plantation age (Age) Olive variety Temperature (Temp) Precipitation (Prec) Age × Variety Age × Temp Age × Prec Variety × Temp Variety × Prec Age × Variety × Temp Age × Variety × Prec

2 1 1 1 2 2 2 1 1 2 2

622.1 201.3 3630.7 3983.8 22.6 6.3 4.8 2.0 0.1 17.3 21.0

b0.001 b0.001 b0.001 b0.001 b0.001 0.002 0.008 0.158 0.800 b0.001 b0.001

18,006 651 3187 6755 13,658 2260 3534 5619 7734 12,625 12,943

b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001 b0.001

precipitation for the two varieties except a slight decrease in variety Blanquette in old plantations. A significant diminution of spore density was recorded with increasing of temperature, except for the variety Blanquette in old plantations. The effects of air temperature and precipitation in interaction with plantation ages and varieties were significant (P b 0.001) (Fig. 4; Table 1). 2. Relationships between mycorrhizal intensity and spore density Linear regressions testing the effect of fungal spore density on olive mycorrhizal intensity indicated that correlations varied by seasons and following plantation ages (for stations studied at the Guelma Basin). In general, seasonal trends of the mycorrhization spore density relationship were similar between the seedlings ‘BK’ and old plantations ‘BL’ (Table 2). The density of spores has a significant effect (SLR: P b 0.001) on the increasing of mycorrhizal intensity within young plantations ‘BB’ in autumn. Similarly, the increase of spore density in winter and spring significantly affected mycorrhization increase in old plantations ‘BL’ and seedlings ‘BK’. However, mycorrhization significantly decreased when spore density recorded in young plantations ‘BB’ decreased (SLR: R2 = 0.67, P b 0.001). Other SLRs were not significant, especially during the summer season in all age classes of olive plantations. 3. Change in mycorrhizal intensity across climate regions Across the climatic gradient in Algeria, mycorrhizal intensity in olive rhizosphere averaged 24.7 ± 11.5% (mean ± SD). The mycorrhizal intensity observed in autumn experienced variations from one climate to another, from one species to another and from one variety to another

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Fig. 3. Effect of air temperature and precipitation on the variation of root mycorrhizal intensity for each plantation age of two olive varieties grown in the basin of Guelma (northern Algeria). The lines represent a linear regression with a GLM (generalized linear model) fit with 95% confidence regions in light grey.

(Fig. 5). The highest values of M% were reached under humid then subhumid climates with respectively 37% and 36% in variety Chemlel and 42% and 39% for variety Rougette. Whereas, M% in the wild-olive was 22% under humid climate and 20% under subhumid conditions. Mycorrhizal levels decreased gradually from desertic to semi-arid climate, reaching 17% for Chemlel, 24% for Rougette and 18% for wild-olive. The semi-arid plantation recorded the lowest mycorrhizal values averaging 12% for the three varieties of olive. The ANOVA showed that M% varied significantly among climate types (F(3, 47) = 62.17, P b 0.001), varieties (F(2, 47) = 29.93, P b 0.001) and the interaction ‘Climate × Variety’ (F(6, 47) = 5.90, P b 0.001). According to Tukey's test, averages of M% were classified into three homogeneous groups of climates: the lowest average was recorded under the semi-arid climate (M% = 13%), followed by M% = 20% recorded in the desertic climate, then by M% recorded in humid (34%)

and subhumid (32%) climates, which were significantly the highest. In addition, Tukey's test indicated three distinct groups according to the factor variety. The wild-olive denoted the lowest value of M% (18 ± 5%), followed by Chemlel with 26 ± 12% and then Rougette with 30 ± 13% (Fig. 5, Fig. 6). The GLM revealed that M% increased with the increase of seasonal rainfall and decreased with increasing temperature. However, only the effect of rainfall was significant (F = 7.45, P = 0.008), while the temperature effect was not significant (Fig. 6, Table 3). 4. Fungal spore biodiversity and variation of MPN The various genera and species identified in the rhizosphere of olive grown under different North African climates showed that there are seven fungal genera Glomus, Septoglomus, Funneliformis, Rhizophagus, Gigaspora, Scutellospora and Entrophospora to which are attached nine

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Fig. 4. Effect of air temperature and precipitation on the variation of spore density in the rhizosphere for each plantation age of two olive varieties grown in the basin of Guelma (northern Algeria). The lines represent a linear regression with a GLM (generalized linear model) fit with 95% confidence regions in light grey.

species (Table 4). These genera were found in different climates except the semi-arid climate that contained only the genera Glomus, Septoglomus and Rhizophagus. Five morphotypes of the genus Glomus were reported in olive plantations at different climatic zones of Algeria. The species Rhizophagus irregularis (syn. Glomus intraradices), Funneliformis mosseae (syn. Glomus mosseae), Septoglomus constrictum (syn. Glomus constrictum), Glomus sp.1 and Glomus sp.3 were the common morphotypes among the four study climates. The morphotype Glomus sp.4 was common between desertic and semi-arid climates. While Glomus sp.2 was common in humid and subhumid stations. Identification of Funneliformis mosseae, Rhizophagus irregularis and Septoglomus constrictum was refined by molecular characterization and other AM fungal spore types could not be identified. Spore densities and MPN of propagules were uneven among climatic regions of the study. The mycorrhizal potential was higher under humid

climate with an average of 2700 propagules, followed by subhumid climate with 1320 propagules. Semi-arid and desertic climates had values of 1015 and 602 propagules, respectively (Table 4). For spore density, the highest value was reported under desertic climate with 320 spores, which gradually diminished to 277 spores under humid climate, 242 under subhumid and 184 under semi-arid climate. But despite these differences, the ANOVA revealed no significant differences between the climates, whether for MPN (ANOVA: F(3, 20) = 1.31, P = 0.302) or spore density (ANOVA: F(3, 8) = 0.59, P = 0.639). The number of propagules and spore density tend to increase from arid to humid, but the variation of this trend was not significant between climate regions (Fig. 7). Indeed, the GLM revealed that regardless of the type of climate, the density of spores significantly increased with the increase of MPN (F = 9.43, P = 0.018), but this increase was the same across sites (F = 3.09, P = 0.098) (Fig. 7).

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Table 2 Statistics of simple linear regressions (SLR) testing the relationships between mycorrhizal intensity (M%) and density of fungal spores following different plantations ages and during different seasons. (R2: multiple R-squared, F: F-statistic, P: P-value). Density of fungal spores in different plantations

Seasons of mycorrhizal intensity Winter

Spring

Summer

Autumn

SLR statistics 2

R F P R2 F P R2 F P R2 F P

Young seedlings ‘BK’ (~1

Young plantation ‘BB’ (6–7

Old plantation ‘BL’ (N100

year)

years)

years)

0.04 5.11 0.026 0.10 12.60 b0.001 b0.01 b0.01 0.959 b0.01 1.00 0.316

0.67 243 b0.001 0.02 3.00 0.086 b0.01 0.05 0.823 0.32 50.40 b0.001

0.11 14.70 b0.001 0.79 455 b0.001 0.01 1.72 0.192 0.03 3.51 0.063

5. Similarity of fungal species richness between climates The Jaccard index calculated for the various climates revealed a 100% affinity between the humid and subhumid climates and 80% between these sites and the desertic climate, followed by 66% of similarity between semi-arid and desertic climates. This degree of affinity was only 50% between semi-arid and humid climates (Table 5).

4. Discussion This study was conducted in ecosystems subjected to Mediterranean climates, with mild and rainy winter, and hot and dry conditions in summer, along a climatic gradient including different climatic variants namely humid, sub-humid, semi-arid and hot desertic. The relationship of root mycorrhizal colonization parameters with environmental factors has attracted considerable attention from researchers that studied this

Fig. 5. Variation of mycorrhizal intensity (mean ± SD) in roots of olive varieties cultivated under different climates of Algeria, North Africa.

in roots of several plant species, but not those of olive which remain very little studied (Udaiyan et al., 1996; Hawkes et al., 2011; Xiang et al., 2014; Neffar et al., 2015). Many environmental factors control the abundance of mycorrhiza at a root system, species composition of AM fungi and their biochemical potential (Datta and Kulkarni, 2012). Among these factors, the edaphic as well as climatic ones are prevailing (Udaiyan et al., 1996; Khade and Rodrigues, 2010); this includes also physicochemical properties of soils (Neffar et al., 2015), the age of host species (Land and Schonbeck, 1991), soil type and different plant communities of the site (Dandan and Zhiwei, 2007), predation and propagule availability (Muthukumar and Udaiyan, 2002). The pattern of mycorrhizal variation in roots of olive trees coincides with the seasonality of the Mediterranean climate. Although Bohrer et al. (2004) and Zhang et al. (2012), stressed the weak influence of abiotic factors on the variation of root colonization that is under the control of plant phenology (Koide and Li, 1990; Bever et al., 1996), highly significant correlations are reported in numerous studies between environmental factors and root colonization (Udaiyan et al., 1996; Muthukumar and Udaiyan, 2002; Neffar et al., 2015). Drought is the main factor behind the decrease of mycorrhizal colonization parameters (Augé, 2001; Hawkes et al., 2011; Panwar et al., 2011). Udaiyan et al. (1996) particularly reported a positive correlation between both spore population and root colonization in Acacia planifrons, and soil moisture, which is linked to rainfall, and a negative correlation between the maximum temperature and root colonization. It appears that climatic factors have a large influence on root colonization (Augé, 2001; Torrecillas et al., 2013). In our study, the highest AM colonization levels were observed during spring and autumn. During these two seasons, alternate periods of moisture and warmth provide most favourable periods for the development of many species of fungi, including mycorrhizae. This seasonal pattern is very similar to the results seen on variety Ferkani of olives grown under different climates in Algeria (Mekahlia et al., 2013). Variably, these peaks can be achieved either in the spring (Bohrer et al., 2004) or in summer (Sigüenza et al., 1996). According to Mandyam and Jumpponen (2008), olive root colonization was higher during spring (period of flowering and vegetative development), than summer (the time of fruit set and fruit growth), and autumn (growth period of fruit and twigs). However, the level of root colonization tends to decline in winter as a period of plant dormancy occurs (López-Sánchez and Honrubia, 1992). It was also reported that root colonization increases were synchronized with the plant growth stages and/or during long periods of stress (Kennedy et al., 2002). The seasonal pattern of root colonization varies significantly depending on the plant species throughout the year, indicating that mycorrhizal symbiosis could be likely considered as specific to the plant species (Ruotsalainen et al., 2002; Muthukumar and Udaiyan, 2002). Our findings indicate that mycorrhization in olive undergoes the combined effect of the phenological stage of the plant and climatic conditions of the habitat. The variation of mycorrhization levels is synchronous with the development cycle of olive that involves two phases: the spring growth that is the largest in the annual life cycle and the autumnal growth. A similar seasonal trend was confirmed by several studies that found that the seasonal variation in AM colonization is related to the phenological stages of plant growth where the highest colonization levels are recorded in spring coinciding with the maximum plant activity (Muthukumar and Udaiyan, 2002; Lingfei et al., 2005; Mekahlia et al., 2013). The climate seasonality of the study sites that is a characteristic of Mediterranean climates, i.e. rains fall principally in autumn and winter (mid-November to February), subsequently the density of spores tends to increase after the rainy season i.e. in the spring. Indeed, Khade and Rodrigues (2010) attest that AM spore density was maximum in April. The abundance of fungi in the dry season was particularly low compared to the wet season (Cregger et al., 2012). Torrecillas et al. (2013) demonstrated that the richness and diversity of AM

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Fig. 6. Effect of air temperature and precipitation on the variation of mycorrhizal intensity in the rhizosphere of olive varieties grown under different climates in Algeria. The lines represent a linear regression with a GLM (generalized linear model) fit with 95% confidence regions in light grey.

communities are positively correlated with rainfall. However, Panwar et al. (2011) report that the AM spore density increases with the increase of soil temperature. Hatimi and Tahrouch (2007) indicate that the production of spores is important during the plant flowering, while it decreases at the end of breeding season when plant activity slowdown with changes in the physiology of roots. The spatial variation of spore density can be associated with physicochemical properties of the soil or maybe spatial climatic variations (Mc Gonigle and Miller, 1996). According to Mc Gee et al. (1997), a density of 5 spores/10 g of soil is capable of initiating a maximum level of root colonization. Spore density may have a positive effect on the level of mycorrhization (Datta and Kulkarni, 2012). This was clearly observed within young olive seedlings and old plantations. The relationship between these two parameters was negative in winter in the young plantations ‘BB’ and positive for the nursery and old orchard where plants need to maintain high and constant mycorrhization during the first stages of their growth and/or at the start of vegetation growth season. Otherwise, it varies during autumn and summer for each plantation age. This relationship does not seem simple, its mechanism and the processes involved remains unknown. This is probably attributed to one side, the sampling method because a strong correlation was found when samples are taken from the same species and the same sites. In contrast, no correlation was found when the samples come from different varieties of the same species or from different sites on the other hand. It could be that the level of spore production does not reflect the abundance of mycorrhizae in roots (Daniell et al., 2001). In addition, the density of spores may be also influenced by growth stages of the plant (Bever et al., 1996; Ji et al., 2010; Tian et al., 2011). According to Udaiyan et al. (1996), this phenomenon seems erratic and these two parameters are not necessarily correlated. As for the variation among climate regions, generally speaking, mycorrhization levels are higher under subhumid and humid climates Table 3 GLM testing the effect of air temperature, precipitation and olive varieties on the variation of mycorrhizal intensity in the rhizosphere of olive orchards cultivated along a climatic gradient in Algeria. (CI: confidential interval, SE: standard error, P: P-value). Parameters

Estimate

2.5% CI

97.5% CI

SE

t-Value

P

Intercept Temperature Precipitation Rougette Wild-olive

14.08 0.02 0.22 4.27 −7.45

−18.85 −1.09 0.06 −1.92 −13.64

47.02 1.13 0.38 10.47 −1.26

16.80 0.57 0.08 3.16 3.16

0.838 0.042 2.730 1.352 −2.357

0.406 0.966 0.008 0.182 0.022

compared to semi-arid and desertic climates, especially in varieties Chemlel and Rougette compared to wild-olive whose M% remains low in all climatic regions. The observed levels of mycorrhization are rather low for a species like olive, which is highly mycorrhiza-dependent (Mekahlia et al., 2013). Mycorrhizal changes between varieties and across the broad climatic gradient in Algeria is probably related to the sampling period. The latter took place in early autumn before the first rains and before the vegetative growth, which both stimulate mycorrhizal symbiosis and trigger colonization of the root cortex. He et al. (2002) reported a positive correlation between AM colonization and soil moisture, which could be an argument that supports our results as the study season was less mesic and because rainfall is an important element of soil moisture. It is believed that mycorrhization is primarily determined by a minimum level of soil moisture to ensure proper functioning of the root system of the olive tree in any climate. It is noteworthy that species responses to mycorrhization are uneven (Udaiyan et al., 1996), and according to our study, it is the same for varieties of the same species.

Table 4 Species richness (S), most probable number (MPN) and spore density of AM fungi identified in olive rhizosphere across a climatic gradient in North Algeria. Study sites AMF spores

Desertic

Semi-arid

Subhumid

Humid

Entrophospora infrequens Rhizophagus irregularis Funneliformis mosseae Glomus sp.1 Glomus sp.2 Glomus sp.3 Glomus sp.4 Septoglomus constrictum Gigaspora sp. Scutellospora sp. Number of genera Species richness (S) MPN

+

−

+

++

+ + + − + + +

+++ + ++ − + + +

+++ ++ +++ ++ ++ − ++

+++ ++ +++ + + − +

+++ ++ 7 9 602 ±

− − 4 6 1015 ±

+ + 7 9 1320 ±

++ + 7 9 2700 ±

213NS 320 ±

421NS 184 ± 99NS

308NS 1389NS 242 ± 146NS 277.66 ±

Spore density [/50 g soil]

135NS

136NS

+++: dominant (N75% of spores), ++: frequent (75–25% of spores), +: present (b25% of spores), –: absent, NS: not significant ANOVA at P = 0.05.

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Fig. 7. Relationship between the density of spores and the MPN under various climate regions in northern Algeria. The lines represent a linear regression with a GLM (generalized linear model) fit with 95% confidence regions in light grey.

Regarding the other two mycorrhizal parameters (MPN and spore density), despite the observed fluctuations, the climate type has no significant effect. However, regardless of the climate region, the density of spores increases with the increase of MPN. Unlike the study of AzcónAguilar et al. (2003) where a negative correlation was registered; although the relationship was positive between the MPN and particularly spores of Septoglomus constrictum, indicating some contribution of fungal spores to the mycorrhizal potential. This fungus is probably better adapted to germinate and colonize roots in all climates. Perhaps it is the same for Glomus that is very frequent in the rhizosphere of olive trees in different climates in Algeria. The values of MPN under different study climates were relatively low, since MPN is considered acceptable around 1500 propagules and low below 500 (Chantelot, 2003). The observed difference can be attributed to contrasts in sporulation ability of different AM fungal species that may induce irregularities in sporal density (Bever et al., 1996). The density of AM spores in soils differs among plant species, these differences may be related to different behaviour of each AM fungal species, even in similar ecosystems (Khakpour and Khara, 2012). Dandan and Zhiwei (2007) indicate that spore density is much influenced by environmental factors. It is known that the olive tree has a strong ability to increase the number of propagules in its rhizosphere (Caravaca et al., 2005).

Table 5 Matrix of Jaccard similarity index (CJ) applied between the four sampled climatic types. Values of richness species are noted above the diagonal whereas index values (%) under the diagonal. Total species richness values are showed in square brackets on the diagonal. Climates

Desertic

Semi-arid

Subhumid

Humid

Desertic Semi-arid Subhumid Humid

[9] 66 80 80

6 [6] 50 50

8 5 [9] 100

8 5 9 [9]

Glomaceae species are the most dominant in the rhizosphere of olive cultivated under different North African climates. These species are known to be widely distributed and is commonly found in different geographical regions worldwide (Stutz et al., 2000). They are well adapted to degraded soils mostly in arid ecosystems where the amount of water and nutrient availability constitute the major constraints for most lifeforms (Bradai et al., 2015a; Bradai et al., 2015b). Glomus species are more adaptable to adjustment of sporulation patterns in varied environmental conditions (Schwarzott et al., 2011). Glomaceae species produce many small-shape spores and are selective of arid environments (Stutz et al., 2000), though AM fungal spore density and species richness can be reduced in degraded soils (Boddington and Dodd, 2000). In the phylogenetic study of Schwarzott et al. (2011), it was concluded that Glomus is the most widely distributed across all terrestrial ecosystems. Its ecological plasticity is due to its high genetic heterogeneity as it can, by itself, constitute a distinct family. It is noteworthy that the structure of AM communities and their distribution are influenced by spatiotemporal variations of climatic and edaphic factors, vegetation cover, plant species, habitat disturbances, cropping practices and sporulation capacity (Dandan and Zhiwei, 2007; Montes-Borrego et al., 2014; Neffar et al., 2015). For example, the various agroecosystems have distinct AM communities (Oehl et al., 2009), with generalist species (Öpik et al., 2006) while other taxa are unique or common to certain habitats (Liu et al., 2011). The comparisons of species diversity between climate regions reveal that there is at least 50% of similarity. These resemblances are clearly represented by the genus Glomus that includes several species with high ecological plasticity (Schwarzott et al., 2011). This feature makes of Glomus the common genus to different climates. According Moreira-Souza et al. (2003), the distribution of AM fungi may be related to soil pH that controls nutrient availability, for example the genus Acaulospora prefers acidic soils (Mukerji et al., 2002). Therefore, we speculate that soil factors play a key role in controlling the pool of AM fungi in soils. In view of that, it is worthwhile to study the effects of soil factors on olive

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mycorrhizal parameters under different climatic regions of North Africa. A focus on edaphic factors of climatic regions with low mycorrhizal parameters may deepen our understanding of mycorrhizal status of one of the most important crop trees in the Mediterranean Agriculture. 5. Conclusion Olive trees grown under North African conditions are dependent of mycorrhiza regardless of the olive variety and age of the plantation. Seasonal dynamics of AM colonization are closely synchronous with phenological stages of plant growth, which are manifested by peaks observed in spring and autumn. These periods correspond to stages of high vegetation activity that also depends on climatic conditions (optimal rainfall and temperature). Mycorrhizal intensity is higher among olive trees planted under humid and subhumid climates compared to desertic and semi-arid climates. The wild-olive is less mycorrhized compared to study olive varieties. The AM fungal community is composed of six genera Glomus, Septoglomus, Funneliformis, Rhizophagus, Gigaspora,

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Scutellospora and Entrophospora. Despite that olive is a species known for its mycotrophic dependence, MPN values of Algerian plantations are low but significantly influence spore density of the soil. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2017.01.049. Authors' contributions AMH, AB and NH designed the study. AMH collected and analysed soil samples, and determined mycorrhizal colonization. HC, SN analysed data and conceived the paper. HC, SN, AMH drafted and revised the manuscript. SG helped with molecular characterization of AM fungal species. All authors read and approved the manuscript.

Acknowledgments The authors warmly thank the associate editor and the seven anonymous reviewers for their constructive comments that greatly improved the quality of the paper.

Appendix 1. Location, climatic and soil characteristics of study olive orchards in northeastern Algeria (North Africa)

Study sites Site features Location Latitude (North) Longitude (East) Altitude (m) Orchard surface area (km2) Climate characteristics Emberger Classification Koeppen Class

Budyko Climate Radiation index of Dryness Budyko Evaporation (mm/year) Budyko Runoff (mm/year) Budyko Evaporation (%) Budyko Runoff (%) Aridity Aridity Index Moisture Index (%) DeMartonne Index Precipitation Deficit (mm/year) Climatic NPPa NPP (Temperature) NPP (Precipitation) Gorczynski Continentality Index Soil variables Soil texture Organic matter (%) pH Understory a

El Oued

Tebessa

Guelma

Bejaia

33°21′11″” 06°47′44″” 60 0.2

35°28′02″” 07°58′09″” 880 0.5

36°37′27″” 07°40′35″” 160 20

36°34′53″” 05°40′05″” 740 10

Desertic BWh B = arid climate W = desert h = hot Desert 22.947 59 0 100 0 Hyperarid 0.04 −96 2 1383 116 2311 116 46.6

Semi-arid BSk B = arid climate S = steppe k = cold Desert 3.438 355 10 97.3 2.7 Semiarid 0.31 −69 15 794 645 1854 645 35.7

Subhumid BSk B = arid climate S = steppe k = cold Steppe 1.895 580 76 88.4 11.6 Dry subhumid 0.64 −36 24 364 1059 2041 1059 22.9

Humid BSk B = arid climate S = steppe k = cold Steppe 1.763 625 94 86.9 13.1 Subhumid 0.66 −34 29 365 1140 1806 1140 0.5

Sand 0.44 7.70 Bare soil

Silty clay 4.09 8.15 Herbaceous

Silt to silty clay 3.26 8.05 Herbaceous

Silty clay 1.39 8.04 Herbaceous

NPP: net primary production in g of dry matter/m2/year is precipitation limited.

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