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Journal of Arid Environments xxx (2017) 1e10

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Journal of Arid Environments

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Changes in arbuscular mycorrhiza fungi spore density and rootcolonization of woody plants in response to exclosure age and slopeposition in the highlands of Tigray, Northern Ethiopia

Emiru Birhane a, b, *, Kahsay Aregawi a, Kidane Giday a

a Department of Land Resources Management and Environmental Protection, Mekelle University, Mekelle, PO Box 231, Ethiopiab Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, PO Box 5003, No-1432 Ås, Norway

a r t i c l e i n f o

Article history:Received 5 July 2016Received in revised form5 January 2017Accepted 2 March 2017Available online xxx

Keywords:DrylandsGrazing landFungiDegradationRestorationSoil properties

* Corresponding author. Department of Land Resouronmental protection, Mekelle University, Mekelle, PO

E-mail address: [email protected] (E. Birh

http://dx.doi.org/10.1016/j.jaridenv.2017.03.0020140-1963/© 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Birhane, E.in response to exclosure age and slope posdx.doi.org/10.1016/j.jaridenv.2017.03.002

a b s t r a c t

The functional link between the aboveground systems and the below ground microorganisms in a plantsystem determines the restoration and re-establishment success of degraded ecosystems. This paperexamined the arbuscular mycorrhiza fungi (AMF) spore density and root colonization in relation to slopeposition and age of exclosures. The first age group had less than five years old, the second age group was5e10 years old and the third age group had 10e15 years old exclosure while the fourth age group hadage between 15 and 20 years. The root and soil samples of 23 plant species that belong to 13 familiesfrom 12 sites of exclosures and grazing land were collected and analyzed using the magnified inter-section and wet sieving method from the highlands of Tigray, Northern Ethiopia. . The AMF root colo-nization ranged from 24% to 96%. The lowest colonization was observed from plant species that belong tothe grazing land, and the highest were from plant roots in exclosures. The spore density was between 30and 2980 of 100 g�1 of dry soil with the lowest from the grazing lands and the highest was fromexclosures of middle slope position. Glomuswas the dominant AMF genus found in all soil from both landuses. A significant difference in spore density (p < 0.05) was observed between slope position and age ofexclosures. AMF root colonization positively correlated (P < 0.05) with spore density. Exclosures ofmiddle slope showed high spore density and root colonization and it increased significantly withincreasing age of exclosures. The presence of abundant AMF spore in exclosures indicated the roleexclosures played in the restoration of ecosystem health. Forestland restoration through exclosures couldfacilitates the survival of planted and regenerated plants by providing enough AMF inoculum in therestored ecosystem. AMF spore density and colonization should be considered as an indicator of resto-ration in measuring success of restoration in the drylands.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

The idea of exclosure involves a protection system, exclusion ofthe degrading agent, to allow the lands to re-establish itselfthrough natural succession process. Exclosures are areas thatexclude human and livestock interference for rehabilitation ofdegraded lands in the drylands (Seyoum et al., 2015). Degradedlands that almost lost their production potentials left for naturebased rehabilitation, and if properly managed and rehabilitatedthrough exclosure system, allow native vegetation to restore. Many

rces Management and Envi-Box 231, Ethiopia.

ane).

, et al., Changes in arbuscularition in the highlands of Tig

case studies conducted in highlands showed that exclosures areeffective in enhancing composition, diversity, and density ofvegetation (Yayneshet et al., 2009). Species that disappeared longtime ago restored following the establishment of exclosures. Forinstance, species that could not be observed for many years in someparts of eastern Tigray, namely Olea europaea subsp. cuspidata andJuniperus procera, reappeared, densities and diversities of the flora,particularly of grasses, and fauna increased, soil erosion decreasedand even dead springs started to flow after exclosures wereestablished (Birhane et al., 2007).

The restoration and re-establishment of degraded ecosystemsshould include not only the aboveground systems but also thebelow ground microorganisms which are linked functionally withplants (Li et al., 2007). The success of any ecosystem restoration

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e102

efforts are likely to depend on the establishment of mycorrhizas,and Arbuscular Mycorrhiza Fungi (AMF) should receive specialconsideration in restoration of degraded ecosystem (Wubet et al.,2003). Owing to the multiple beneficial effects on plant perfor-mance and soil health, AMF are essential for the restoration and re-establishment of the vegetation in degraded ecosystems (Dhillionand Gardsjord, 2004). AMF are more commonly distributed thanother types of mycorrhizal associations (Smith and Read, 2008) andare keystone organisms that form an interface between soils andplant roots, and are sensitive to changes in soil and plantenvironment.

Disturbance can affect the incidence of AM fungi in both agri-cultural and natural ecosystems. Land use change and/or distur-bance of soil can reduce mycorrhizal infection and several factorsmay be responsible. Soil disturbance negatively affect the func-tionality of AMF (Trejo et al., 2016). There may be effects of tillageon root growth affecting the degree of root colonization bymycorrhizal fungi (Borie et al., 2006). The influence of grazing onsoil nutrient availability and host plant productivity may causeinconsistent effects on AMF community composition and structure(Bai et al., 2013). Grazing of pasture grasses in the field has beenfound to affect the amount of root length infected by decreasingroot length per unit volume of soil (Yang et al., 2013). Grazers alsoinfluence allocation to AMF morphological structures by changingsoil nutrient condition through direct inputs of N and P in dung andurine deposition (van der Waal et al., 2011; Schnyder et al., 2010).AMF differ in their response to the mineral environment of the soil(Brundrett, 2004).

One way by which plants can potentially enlarge ecosystemproductivity and stability is by forming mycorrhizal associations(Eriksson, 2001). Plants are most likely to form associations withand benefit from mycorrhizal fungi under situations in whichavailability of one or more soil nutrients, including water, is low(Smith and Read, 2008). Tropical savanna soils have been erodedand poor in nutrients resulting to reduced plant productivity(Pimentel, 2006). AMF are of particular significance to the plant insoils that are nutrient poor (Jeffries et al., 2003). Moreover, AMFserve as sensitive indicators of ecological soil quality if theyrespond to environmental variation in a predictable way(Verbruggen et al., 2012). Information about species composition ofAMF community appears important to recognize mycorrhizalfunction in the ecosystems. It is evident that AMF are essential forthe functioning of terrestrial ecosystems. Therefore, understandingthe impact of land use on AMF abundance in tropical soils isimportant. Forest restoration in protected exclosures has become awidespread practice to fight land degradation in the highlands ofNorthern Ethiopia (Mekuria and Aynekulu, 2013). Exclosures havebeen implemented in grazing areas for the past decades in Ethiopiaand have been effective in regenerating natural vegetation, con-trolling soil erosion and increasing soil fertility (Baudron et al.,2015). Despite this effort, studies that evaluate the effectivenessof different exclosure habitat types to restore degraded soils andvegetation, their role to the enhancement of microorganisms insoils are lacking. Besides, it is hardly possible to find studies thatevaluate the benefits of exclosures to restore arbuscular mycorrhizafungi (AMF). This paper aimed at investigating the spore densityand root colonization of AMF in relation to land use, age and slopepositions of the exclosures and the dynamic with soil nutrients. Theresearch questions answered in this paper were; is conversion offree grazing land to exclosure increased AMF spore density inrhizosphere soils and colonization of plant roots? Does age ofexclosures at different elevation gradient influence the availabilityof AMF spore density and percent of root colonization? What is therelationship of AMF spore density and root colonization in relationto the available nutrients?

Please cite this article in press as: Birhane, E., et al., Changes in arbuscularin response to exclosure age and slope position in the highlands of Tigdx.doi.org/10.1016/j.jaridenv.2017.03.002

2. Materials and methods

2.1. Study area

The study was conducted in the highlands of Tigray region,Northern Ethiopia in four zones and four districts (Fig. 1). Therewere 12 sites having exclosures and a grazing land. Exclosure is amethod of rehabilitating land by protecting an area from theinterference of animals and human encroachment for limitedperiod of time, depending on site capacity and vegetation re-establishment (Seyoum et al., 2015). The grazing lands are areasopen for grazing continuously by livestock. The exclosures weredivided in to four age classes and three slope positions to study theage, slope position and land use effect on AMF and soil physico-chemical properties. The first age group had less than five yearsold exclosure with triplicates sites, the second age group was 5e10years old exclosure with triplicates sites and the third age grouphad 10e15 years old exclosure while the fourth age group had agebetween 15 and 20 years. All sites have tropical semi-arid climate.The altitude of the study sites ranged from 2232 to 2937m.a.s.l. Therainy season usually occurs between June and September (Fig. 2)and the growing season varies between 90 and 120 days. Thehighest rainfall is in July and August that ranges between 162 and228 mm. The maximum temperature is in the months of May andJune (Fig. 2).

Luvisols (Alfisols), Regosols (Entisols), Cambisols (Inceptisols)and Calcisols (Aridisols) were major soil groups in the study area(WRB, 2006). The study sites were dominanted by Luvisols (Alfi-sols) and Cambisols (Inceptisols). Acacia etbaica, Acacia seyal (Del.),Becium grandiflorum (Lam.) Pichi-Serm. Euclea racemosa subsp.schimperi (A. DC.) Dandy and Maytenus arbutifolia (Hochst. ex. A.Rich) Wilczek were the common woody vegetation species inexclosures and grazing lands (Mekuria and Aynekulu, 2013). Theexclosures are mainly covered by trees, shrubs and the ground bygrass. The life form of woody plants in the exclosures were 35.1%trees and 39.73% were shrubs while the rest were woody herbs andclimbers. The life forms in the open grazing lands were 83.37%shrubs and 5.7% were trees while the rest were woody herbs;shrubs significantly outnumbered the trees in the exclosures. Theabundance of the naturally regenerated woody plants in theexclosures was 91.03% while 8.9% was found artificially planted butno planted seedling was observed in the open grazing lands. Theabundant species in the exclosures was composed of naturally re-generated species.

Mixed farming system that integrates crop and livestockwas themain means of livelihood. The major land uses were cultivated land(9e33%), forestland (3e58%), exclosure (3e16%), communal graz-ing land (6e39%) and others (20e41%) of the total area (Mekuriaand Aynekulu, 2013). The main crops cultivated were Teff (Era-grostis teff (Zucc.) Trotter), Bread wheat (Triticum aestivum), Maize(Zea mays L.), Sorghum (Sorghum bicolor), Barley (Hordeum vulgare),and Faba bean (Vicia faba).

2.2. Experimental design

The role of exclosure on soil physico chemical properties, AMFspore density and root colonization, were studied by takingrhizosphere soils and roots under the different slope positions andages of exclosures and adjacent grazing lands as controls. Theassumption were prior to establishment of exclosures, the controlsites had similar conditions as the exclosures were established onthe same communal grazing lands which were used for livestockgrazing (Mekuria and Aynekulu, 2013). The experiment wascomposed of 12 experimental units with four treatments (agegroups) replicated three times in the two land uses. In the entire

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

Fig. 1. Location of the study sites in the highlands of Tigray, North Ethiopia.

Fig. 2. Monthly average rainfall and temperature of the study area from 2002 to 2014(EMA, 2015).

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e10 3

study 216 plots (12*3 plots * three landscape positions *an exclo-sure and adjacent grazing land as a control) were examined. In eachexclosure and grazing land three transects spaced at a minimumdistance of 75 mwere randomly established (Fig. 3). The number oftransects were based on vegetation density, spatial heterogeneityof vegetation, and area of the site (Mekuria and Aynekulu, 2013).The first transect laid 30e50 m inside the exclosures and grazinglands to avoid edge effects. Transects were parallel to each otherand to the topography of the landscape. In each transect three

Please cite this article in press as: Birhane, E., et al., Changes in arbuscularin response to exclosure age and slope position in the highlands of Tigdx.doi.org/10.1016/j.jaridenv.2017.03.002

landscape positions/elevation gradient (upper slope, mid slope andfoot slope) were delineated and in each landscape position asampling plot measuring 5 m � 5 m were established for soilphysical and chemical analysis (Fig. 3). The upper slope (US) posi-tion is the uppermost portion of each study site and it can receivelittle or no overland flow but may contribute runoff to down slopeareas. The middle slope (MS) position receives overland flow fromthe upper slope and contributes runoff to the foot slope (FS). The FSrepresents the lowest part of each study site and receives overlandflow from both mid and upper slopes.

2.3. Plant and soil sampling methods

The woody plant species and the associated rhizosphere soilssampled from August 2015 to September 2015. A total of 507 rootand soil samples were collected from the four sides of each woodyplant found in 5 m*5 m plots found in all exclosures and grazinglands to enumerate spore abundance and root colonization. Theidentity of the woody plant was identified at the field and verifiedusing reference books (Inga et al., 2003).

2.3.1. AMF colonization and spore density enumerationThe collected fine roots were put into beaker and carefully

washed with tap water until the soil was removed and became freeof any soil particles. Fine root samples put into plastic jar and filledwith 97% ethanol to preserve the roots until they were ready forprocessing. AMF spores separated from the soil by the wet sievingand 50% sucrose centrifugation method (Brundrett et al., 1996).

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e104

Spores counted from 25 g soil using dissecting microscope with100X magnification power. Spores grouped into genera of differentAMF according to the number of morphological characteristics suchas; spore size, shape, color, wall structure and hyphal attachment(simple, swollen or bulbous). To identify root colonization, the rootsfirst washed to remove ethanol, cut into 1e2 cm length and insertinto the heat resistant bottle containing 10% KOH solution, thenautoclaved for 20 min at 120�c.The roots washed to remove KOHand put into 10% H2O2 for further bleaching and clearing for about15min. Cleared roots captured on a fine sieve and rinsedwithwaterbefore transferring them into the HCl solution. Roots were acidifiedwith 3% HCl for about 30 min, and stained in trypan blue (0.05% in5:1:1 lactic acid: glycerol: distilled water) over night. The stainedroots washed and added to 50% glycerol for destining and preser-ving until further processing. Then roots were prepared on slidelengthwise by selecting 20 subsample roots. The proportional rootcolonization by AMF estimated using the magnified intersectionmethod with hairline graticule inserted into an eyepiece acted asthe line of intersection with each root at 400-magnification powerunder the compound microscope. Percentage of root length colo-nization (percentage RLC) calculated from 100 or more in-tersections for each root sample.

2.3.2. Soil sampling and analysisThe soil samples for one plot was taken from the four corners

and the center of a square plot at 50 cm soil depths in order todetermine soil organic carbon, pH, EC, N, K and P in exclosures andgrazing lands from a total of 216 soil samples. The soil samples weremixed andmake a single composite sample to represent the sampleplot. A 1 kg composite soil sample taken and put into plastic bags,secured, labeled and brought to the soil laboratory. Soil sampleswere analyzed for pH and electrical conductivity on 1:2.5, soil:water suspension method. Organic matter was analyzed using theWalkley-Black method (Ranst Van et al., 1999), total nitrogen con-tent by the Kjeldahl method (Bremmer and Mulvaney, 1982).Texture was analyzed with hydrometer method (Gee and Bauder,1982). Available P determined using the Olson method (Olsen andSommers, 1982).

Fig. 3. Experimental designs of the soil and plant sampling in exclosure and gr

Please cite this article in press as: Birhane, E., et al., Changes in arbuscularin response to exclosure age and slope position in the highlands of Tigdx.doi.org/10.1016/j.jaridenv.2017.03.002

2.3.3. Statistical analysisThe differences in spore density, root colonization and soil

physico chemical properties between an exclosure and thecommunal grazing land at different age groups and landscape po-sition were analyzed using ANOVA with Tukey HSD test afterchecking normality test using SPSS version 20 software. Pearsoncorrelation test used to analyze the relationships between abun-dance of spores and root colonization and soil physico-chemicalproperties.

3. Result

3.1. Woody plants AMF spore abundance in exclosure and grazingland

Land use type, slope and age were the main source of variationfor significant difference in AMF spore density (Table 1). Theinteraction effect land use *slope, land use *age was significantlydifferent (P < 0.05), but the interaction effect among slope*age andland use*age*slope was not significantly different (Table 1).

AMF spore density ranged from 40 spores 100 g�1 to 2980spores 100 g�1 of dry soil in exclosures and 30 spores 100 g�1 to2200 spores 100 g�1 of dry soil in grazing lands. The highest sporenumber observed from the middle slope position in exclosure andthe lowest was under grazing land. There was a significant differ-ence in spore density (p < 0.05) between exclosures and grazinglands. Glomus, Acaulospora, Scutellospora, Gigaspora and SclerocystisAMF genus were identified (Table 2).

Glomus was the dominant genera followed by Acaulospora,Scutellospora, Gigaspora and sclerocytis respectively (Table 2). Theaverage Glomus density in exclosures was 38.5% greater than theGlomus found in grazing land of the study sites. Within the exclo-sures glomus was 49.81%, 76.03%, 90.89% and 95.12% greater thanAcaulospora, scutellospora, gigaspora and sclerocytis respectively.The mean spore density in the middle slope position was 37.39%and 47.69% greater than from themean spore densities found in theupper and foot slope positions respectively. A significant difference(F 5.15, p < 0.05) in spore density was observed among the slopepositions (Fig. 4).

The maximum and minimum spore density among the age

azing land in the upper slope (US), middle slope (MS) and foot slope (FS).

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e10 5

groups was (985, 30), (756, 11), (2010, 40) and (1211, 50) for the agegroups less than five, 5e10, 10e15 and 15e20 respectively. Withincreasing age of exclosures, the spore density has shown incre-ment in all age groups (Fig. 4). A significant difference in AMF sporedensity (P < 0.05) was observed between the different age groupsof exclosures (Fig. 5). Old exclosures had significantly high sporedensity (Fig. 5).

AMF spore density was consistently and significantly higher inthe middle slopes from all age groups (Fig. 6). The middle slopefrom the 15e20 year old exclosures had higher spore density fol-lowed by the 10 to 15 old exclosure and 5e10 year old exclosure.The lowest spore density was from the foot slopes of youngerexclosures followed by the age group between 5 and 10 years old.

The highest spore density observed from Juniperus proceraspecies in the family cupressaceae followed by Olea europaea in thefamily oleaceae and Pittosporum viridiflorum species in the familypittosporaceae (Table 3). Acacia etbaica, Senna singueana and Crotonmacrostachyus had low spore abundance (Table 3). Keystoneindigenous species (Juniperus procera and Olea europaea) hadhigher number of AMF spore abundance. The AMF spore densitywas comparable in the species from the Fabaceae family (Table 3).

3.2. AMF root colonization from woody plants in exclosure andgrazing land

Land use significantly affected AMF root colonization (Table 4).AMF root colonization was significantly higher in exclosurescompared to the grazing land (P < 0.05). There was a significantdifference in root colonization among the land uses, slope and ageof the exclosures where the tree roots excavated (Table 4). The treespecies in the middle slope had higher root colonization followedby the upper slope and foot slope. Exclosures between the ages of10e20 year had higher hyphal colonization than the younger andoldest exclosures (Table 4). The interaction between land use andage; and land use and slope was significant (p < 0.05) while theinteraction effect age*slope and land use*age*slope was notsignificantly different (P > 0.05). The highest percentage of AMFwas HC (96%) followed by MHC (94%), AC (64%), VC (53%) inexclosures. In the grazing land, the highest AMF was HC (61%) andthe lowest was VC (24%).

All the 23 plant species showed mycorrhizal colonization(Table 5). There was a significant difference (P < 0.05) in rootcolonization among the studied tree species. Typical structures likehyphae, arbuscules and vesicles observed in all tree species.Increased spore density shows increased root colonization in thetwo land use types and vice versa (Table 6), with a positive corre-lation (r ¼ 0.21) between spore density and percent of root colo-nization. However, species that had higher spore density did notshow higher colonization except Pittosporum viridiflorum that wasthe third highest in spore density. Species in the Fabaceae familyshowed a consistent and comparable mycorrhizal root colonizationsimilar to spore density. There was no clear difference in AMF spore

Table 1ANOVA for spore density in exclosure and grazing land with slope positions anddifferent age groups.

Source F P

Land use 4.469 0.035Slope 4.152 0.016Age 17.51 0.00Land use*slope 0.369 0.042Land use*age 2.584 0.043Slope*age 0.577 0.749Land use*age*slope 0.159 0.987

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density and root colonization between evergreen and deciduousspecies and between trees and shrubs (Tables 3 and 5).

3.3. Availability of soil nutrients and AMF

Soil chemical properties at older age exclosure had better pH,EC, P, N, K, OC and OM than the younger age groups of exclosures(Table 6). Availability of soil chemical properties decreased withdecreasing age of exclosure (Table 6). The mean pH of the two landuse types lies between 7.13 and 7.32 which can be categorized asneutral and it was not significant (P > 0.05). The available phos-phorus found in grazing land was 37% higher than the availablephosphorus in exclosures (Table 6). The available phosphorus foundin foot slope was 39% and 42% greater than the upper and middleslope positions respectively. Unlike the available phosphorus,percent of organic carbon and percent of organic matter was 20.8%and 18.4% greater in exclosures respectively compared with thegrazing lands. The upper sloped had higher OC and OM (Table 6).

Spore density was highly significantly correlated with EC(P < 0.001) and was significant with soil pH (P < 0.05). The corre-lation between spore density and available phosphorus was notsignificant (Table 7). Organic carbon, organic matter, total nitrogenand available potassium did not show significant correlation withAMF spore density (Table 7).

4. Discussion

Arbuscular mycorrhiza fungi were present in all woody plantroots and rhizosphere soil samples. There was a significant varia-tion in the abundance of AMF spores in the rhizosphere soil of treespecies and their families. The difference in AMF spore densitybetween samples could be due to the difference in micro climaticand edaphic properties, spatial and temporal variation, vegetation,and host specificity between fungi and woody plants, age of thehost plants, disturbance, and differential sporulation ability of AMFtaxa (Husband et al., 2002). Soils from closed area had high sporenumbers followed by grazing area (Covacevich and Berbera, 2011).Soil disturbance and land uses negatively affect AMF spore densityand functionality (Trejo et al., 2016). The higher spore abundance inwoody plants from the exclosures indicated the negative conse-quence of disturbance on AMF spore density. This has also been thecase (Songachan et al., 2011; Birhane et al., 2010) where manage-ment in the form of exclosure had a positive effect on sporeabundance. The spore density in undisturbed land was significantlyhigher than the cropped land, which supported the view that dis-turbances reduce AMF spore density (Ferrol et al., 2004; Oehl et al.,2003). The AMF spore density found in this study is comparable toother studies in the tropical area and is higher than spore densitiesfound in degraded drylands. The spore density was 110e2600spores 100 g�1 dry soil in tropical forest and pasture (Picone, 2000)and 5 to 6400 spores 100 g�1 dry soil in a valley savanna of the drytropics (Tao et al., 2004). The spore density from the dry deciduouswoodlands of northern Ethiopia associated with different acaciaspecies was 11e32 spores 100 g�1 dry soil (Birhane et al., 2010), 49to 67 spores 100 g�1 dry soil in India (Lakshman et al., 2001).Availability and activity of Arbuscular mycorrhiza fungi spores canbe affected by vegetation removal (Boddington and Dodd, 2000)resulting to a significant decrease in spore density and colonization.The presence or absence of host plants and the plant species beinggrown affects AMF survival in the soil (Wiseman and Wells, 2005).Generally disturbed ecosystems show a decrease in spore abun-dance (Trejo et al., 2016; Hailemariam et al., 2013).

In the present study, Glomus and Acaulospora were the domi-nant genus. The possible reasons for the predominance of Glomus isthat spores of Glomus species have different adaptability and have

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

Table 2Woody plant AMF spore density of the genera across land use, slope position and age groups.

Source AMF spore genera

Glomus Acaulospora scutellospora Gigaspora Sclerocytis

LU EX 13.7 ± 0.48a 6.8 ± 0.28a 3.3 ± 0.14a 1.2 ± 0.08a 0.6 ± 0.71a

GL 8.4 ± 0.35b 4.2 ± 0.19b 2.0 ± 0.1b 0.7 ± 0.05b 0.3 ± 0.62b

AGE <5 4.6 ± 0.28a 2.7 ± 0.19a 1.5 ± 0.13a 0.6 ± 0.09a 0.5 ± 0.06a

5e10 10.4 ± 0.57b 5.9 ± 0.38b 2.9 ± 0.23b 1.2 ± 0.15b 0.6 ± 0.08b

10e15 17.8 ± 0.62c 7.7 ± 0.6c 3.7 ± 0.29c 1.2 ± 0.17c 0.6 ± 0.08c

15e20 21.6 ± 0.59d 10.8 ± 0.47d 4.8 ± 0.29d 1.8 ± 0.22d 0.8 ± 0.09d

Slope US 9.9 ± 0.42a 5.2 ± 0.26a 2.5 ± 0.14a 1.0 ± 0.09a 0.5 ± 0.05a

MS 15.8 ± 0.63b 8.0 ± 0.37b 3.8 ± 0.18b 1.4 ± 0.11b 0.6 ± 0.05b

FS 8.3 ± 0.47c 3.7 ± 0.23c 1.8 ± 0.12c 0.6 ± 0.06c 0.3 ± 0.04c

Means in the same column followed by same letter do not differ significantly at P < 0.05, mean ± SEM. LU: Land use, EX: Exclosures, GL: grazing land, US: upper slope, MS:middle slope, FS: foot slope.

Fig. 4. Arbuscular mycorrhiza spore density in relation to slope position. White bar forupper slope position, dotted bar for middle slope position, and bold dotted bars forfoot slope position. Different letters above the bars indicate significant differencesbetween the samples. Bars indicate mean ± SEM.

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e106

high plasticity to both biotic and abiotic factors (Dare et al., 2013).Some species of Glomus even found in intensively disturbed landuses such as farmlands with frequent tillage (Turrini et al., 2010;Wang et al., 2008) which supports the comparable presence ofGlomus in the open grazing lands in the present study. GeneralistAMF species that adapt to wide environmental variables areimportant in the restoration of degraded lands (An et al., 2008) andit can be easily applied and scaled up to wider range of restorationactivities.

Large variations in spore density between slope positions aremainly due to the properties of the soils, host relations, and thedifferential survival strategies of AMF. The highest spore numberfound from middle slope of exclosure. This finding is in agreementwith Carvalho et al. (2012) the total number of spores were low inlow altitude compared to the high altitude. The correlation be-tween altitude and AMF spore density was low (r2 ¼ 0.17). Theoccurrence of common species at the high altitude and lowerspecies competition at low altitude could influence the AMF sporedensity at different slope gradient (Carvalho et al., 2012). Our resultis in contrast to spore abundance during the rainy season. Higher

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spore density is expected at the foot slopes as the spores can movedownslope following the gradient. In the absence of rainfall,accumulation of spore decrease at the lower elevation as far asthere are no spores coming down with rainwater (Habiyaremyeet al., 2015). The lower elevation sites in an altitudinal gradientsupport higher richness of AMF and the mechanisms responsiblefor the trend could be an increase in temperature and number ofpotential hosts (Lugo et al., 2008).

Age of exclosures had a significant impact on spore density. Thisresult is in line with Covacevich and Berbera (2011), indigenousarbuscular mycorrhizae increase with increase in successionalstages in a tropical dry forest biome. A plant re-establishment studyusing a mixture of pioneer, secondary and climax plant speciesfound that the rhizosphere of pioneer species had the lowestnumber of spores, and this increased gradually until the occurrenceof climax species and concluded that the number of AMF sporesincreases with the stage of succession (Carrenho et al. 2001). Stableecosystems composed of climax species and undisturbed sites insemiarid fields showed higher mycorrhizal sporulation (Paganoet al., 2010). The increase in the age of restoration increasesecosystem stability that favors spore density and AMF sporulationthrough establishing permanent hyphal networks. This studyconfirmed AMF spore density after conversion of free grazing landto exclosure greatly vary with age of exclosures, as the age ofexclosure increases spore density also increases.

There was a positive change in AMF root colonization when freegrazing lands converted to exclosures. The highest percentage(96%) of AMF root colonization was in exclosure while the lowest(24%) was in grazing land. Higher colonization observed from soilsof less disturbed sites that is related to the species composition ofeach soil and their ability to promote plant growth (Trejo et al.,2016). The percentage of colonization of AMF in the roots ofwoody plants differ from locality to locality (Egbert and Mary,2009) and is highly affected by disturbance. The result of thisstudy indicated that soil disturbance or land use change negativelyaffect the colonization of woody plant roots by AMF. The freegrazing land is continuously grazed by livestock and couldcontribute to the disintegration of the AMF hyphae. Grazing ofpasture grasses in the field has been found to affect the amount ofroot length infected by decreasing root length per unit volume ofsoil (Yang et al., 2013). Reducing soil disturbance and increasevegetation cover are important factors to preserve the benefits thatAMF can provide to restore ecosystem (Smith et al., 2011).

In the present study, all the collected woody plant species weremycorrhizal, but colonization percentage and spore density greatlydiffers within the land uses of different elevation gradients and agegroups of exclosures. As the spores represent the dormant state ofthe fungus, the physiologically active state is most likely the keyfactor of the seasonal spore counts. This factor also can effect on

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

Fig. 5. Arbuscular mycorrhiza fungi spore abundance in rhizosphere soil of different age groups. Different letters above the bars indicate significant differences between thesamples. Bars indicate mean ± SEM.

Fig. 6. Arbuscular mycorhiza fungi spore density from different age groups and slope position of exclosures. White bars foot slope, dotted bars middle slope, and bold dotted barsfor upper slope. Bars indicate mean ± SEM.

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e10 7

spore number estimation as well as species diversity (Muthukumaret al., 2003). The relationship between AMF spore density andpercentage of woody plant root colonization are influenced bymany biotic and abiotic environmental factors. Our study revealedthat, increased spore density shows increased colonization in thetwo land use types and vice versa. However, some researchersrecommended that no significant correlation between AM

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colonization and spore density (Li et al., 2007). Generally, AMFcolonization is influenced by spore availability (Muthukumar et al.,2003). The irregular spatial distribution of AMF spores and thecomplex structure of the underground root component should beconsidered as most important factors affecting AMF spore densitythat could contribute to variable rates of AMF colonization amongplants (Zhao et al., 2001).

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

Table 3AMF spore density for woody plant species and their respective family 100 g�1 of dry soil.

Plant species Family Spore 100g�1 Plant species Family Spore 100g�1

Abutilon longicuspe Malvaceae 328 ± 8.4 Euclea racemosa Ebanaceae 276.3 ± 23.9Acacia etbaica Fabaceae 248.4 ± 36.1 Juniperus procera cupressaceae 683.5 ± 74.8Acacia lahai Fabaceae 440.0 ± 47.2 Maytenus arbutifolia celastraceae 475.0 ± 46.4Acacia saligna Fabaceae 318.8 ± 82.4 Maytenus senegalensis celastraceae 392.5 ± 79.9Acacia seyal Fabaceae 430.5 ± 49.8 Olea europaea oleaceae 617.7 ± 302.8Acokanthera schimpri Apocynaceae 340.0 ± 92.5 Osyris quadripartita loranthaceae 405.0 ± 68.8Buddleja polystachya Loganiaceae 330 Pittosporum viridiflorum pittosporaceae 610Calpurinia aurea Fabaceae 405.7 ± 70.1 Rhus glutinosa Anacardiaceae 295.6 ± 41.5Carissa spinarum Apocynaceae 404.5 ± 76.3 Rhus retinorrhoea Anacardiaceae 323.3 ± 70.5Croton macrostachyus Euphorbiaceae 251.4 ± 63.0 Rosa abyssinica Rosaceae 300Dichrostachys cinerea Fabaceae 340.0 ± 170.0 Senna singueana Fabaceae 249.3 ± 35.2Dodonaea angustifolia Sapindaceae 393.7 ± 77.2

Table 4Root colonization in exclosure and grazing land across the age groups and slope positions.

Source Root colonization (%)

HC MHC VC AC

LU EX 77.4 ± 0.39a 74.5 ± 0.41a 36.7 ± 0.36a 45.2 ± 0.42a

GL 75.9 ± 0.44b 73.3 ± 0.46b 36.6 ± 0.37b 43.9 ± 0.46b

Age <5 75.2 ± 0.69a 71.9 ± 0.71a 45.5 ± 0.55a 37.2 ± 0.67a

10e5 78.5 ± 0.82b 75.1 ± 0.88b 42.6 ± 0.77b 35.5 ± 0.76b

15e20 78.7 ± 0.97c 76.3 ± 0.97c 46.6 ± 1.09c 38.0 ± 0.79c

>20 77.4 ± 0.67d 75.1 ± 0.67d 46.2 ± 0.87d 36.4 ± 0.65d

Slope US 76.7 ± 0.52a 74.0 ± 0.54a 45.3 ± 0.52a 36.4 ± 0.43a

MS 76.8 ± 0.52b 73.9 ± 0.55b 44.1 ± 0.55b 36.8 ± 0.43b

FS 76.07 ± 0.49c 74.1 ± 0.5c 44.5 ± 0.57c 36.8 ± 0.47c

LU*age 133.4 ± 44.49a 128.3 ± 42.79a 125.4 ± 41.82a 72.3 ± 24.12a

LU*slope 285.8 ± 142.93b 433.7 ± 216.88b 174.8 ± 87.4b 69.8 ± 34.9b

Age*slope 100.7 ± 16.78c 235.5 ± 39.26c 56.9 ± 9.49c 65.0 ± 10.84c

LU*age*slope 133.9 ± 22.32c 175.3 ± 29.22c 115.7 ± 19.28c 388.2 ± 64.71c

Means in the same column followed by same letter do not differ significantly at P < 0.05, mean ± SEM. LU: Land use, EX: Exclosures, GL: grazing land, US: upper slope, MS:middle slope, FS: foot slope. HC, MHC, VC and AC are percentage of root length with hyphal, mycorrhiza hyphal, vesicles and arbuscule colonization, respectively.

Table 5Mean percent of root colonization of tree species and their respective families in exclosure and adjacent grazing land.

Plant species Root colonization (%)

Family %HC %MHC %VC %AC

Abutilon longicuspe Malvaceae 82 ± 3.28 79.4 ± 3.07 39.6 ± 2.01 45.4 ± 4.2Acacia etbaica Fabaceae 74.8 ± 0.8 72.4 ± 0.85 38.0 ± 0.68 47.2 ± 0.81Acacia lahai Fabaceae 79 ± 4.6 76 ± 5.85 33.3 ± 2.02 41 ± 4.16Acacia saligna Fabaceae 77.8 ± 0.9 74.5 ± 1.43 36.3 ± 1.53 47.7 ± 2.87Acacia seyal Fabaceae 78.3 ± 0.72 75.3 ± 0.75 37.5 ± 0.7 44.2 ± 0.76Acokanthera schimpri Apocynaceae 77 ± 2.44 74 ± 2 38 ± 1.34 48.6 ± 2.71Buddleja polystachya Loganiaceae 79 75 38 42Calpurinia aurea Fabaceae 76.1 ± 0.98 73.6 ± 1.12 35.8 ± 1 42.4 ± 1.29Carissa spinarum Apocynaceae 79.9 ± 1.5 76.7 ± 1.55 37.6 ± 1.35 43.5 ± 1.43Croton macrostachyus Euphorbiaceae 85.2 ± 2.5 82.1 ± 2.69 41.3 ± 1.88 46.1 ± 1.71Dichrostachys cinerea Fabaceae 79 ± 5.85 76.3 ± 6.76 37 ± 3.6 45.3 ± 5.45Dodonaea angustifolia Sapindaceae 77.4 ± 1.2 74.1 ± 1.17 36.4 ± 1.33 48.4 ± 1.31Euclea racemosa Ebenaceae 74.4 ± 0.96 72.4 ± 1.02 36.5 ± 0.75 46.5 ± 0.91Juniperus procera Cupressaceae 77.3 ± 0.63 73.8 ± 0.62 35.5 ± 0.63 42.2 ± 0.7Maytenus arbutifolia Celastraceae 76.7 ± 0.75 74.1 ± 0.86 35.8 ± 0.73 42.3 ± 0.81Maytenus senegalensis Celastraceae 75.9 ± 1.94 74 ± 2.1 35 ± 1.67 39.6 ± 2.54Olea europaea Oleaceae 77 ± 1.6 75.1 ± 1.54 34.6 ± 1.65 47.5 ± 2.47Osyris quadripartita Loranthaceae 75.1 ± 2.25 74.3 ± 2.74 39.1 ± 3.47 46.7 ± 1.95Pittosporum viridiflorum Pittosporaceae 89 89 44 61Rhus glutinosa Anacardiaceae 76.3 ± 1.66 73 ± 1.74 35.2 ± 1.35 42.3 ± 3.71Rhus retinorrhoea Anacardiaceae 75.6 ± 5.36 72 ± 3.31 31.3 ± 1.85 44.4 ± 2.51Rosa abyssinica Rosaceae 60 60 35 40Senna singueana Fabaceae 75.6 ± 2.24 72.6 ± 2.24 38.1 ± 1.74 44.6 ± 0.31

HC, MHC, VC and AC are percentage of root length with hyphal, mycorrhiza hyphal, vesicles and arbuscule colonization, respectively.

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e108

Availability of soil chemical properties decreased withdecreasing age of exclosure (Table 6). Grazing impacts on soilproperties depends on grazing intensity, where higher values of pH,

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available P, and Mg found in closed area (Ajorlo et al., 2011). Soilchemical properties of exclosures were significantly higher thangrazing land (Mekuria and Aynekulu, 2013). Our data shows soil pH

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

Table 6Soil properties in exclosures and grazing lands with increasing age of exclosures and slope gradient.

pH EC(ms/m) P (ppm) OC (%) OM (%) TN (%) K (ppm)

LU EX 7.13 ± 0.17a 0.094 ± 0.01a 2.7 ± 0.5a 2.4 ± 0.2a 4.13 ± 0.34a 0.13 ± 0.15a 165.73 ± 30.2a

GL 7.32 ± 0.15a 0.1 ± 0.19b 4.37 ± 1.9b 1.9 ± 0.17b 3.37 ± 0.29b 0.15 ± 0.02b 231 ± 71.15b

Slope US 7.19 ± 0.19a 0.11 ± 0.03a 2.98 ± 0.9a 2.3 ± 0.23a 4.06 ± 0.4a 0.155 ± 0.02a 149.87 ± 32.2a

MS 7.09 ± 0.19a 0.08 ± 0.01b 2.83 ± 0.8b 2.08 ± 0.19b 3.59 ± 0.33b 0.155 ± 0.06b 191.75 ± 54b

FS 7.4 ± 0.21a 0.09 ± 0.01c 4.9 ± 2.9c 2.1 ± 0.3c 3.63 ± 0.52c 0.12 ± 0.02c 254.5 ± 101.3c

Age <5 7.11 ± 0.12a 0.02 ± 0.2a 2.21 ± 0.3a 1.3 ± 0.1a 2.11 ± 0.21a 0.11 ± 0.01a 98.6 ± 21.4a

10e15 7.21 ± 0.43a 0.031 ± 0.18b 4.64 ± 0.9b 1.8 ± 0.11b 2.71 ± 0.27b 0.19 ± 0.08b 127 ± 28.6b

15e20 7.3 ± 0.32a 0.05 ± 0.23c 5.14 ± 0.7c 2.3 ± 0.14c 3.2 ± 0.3c 0.21 ± 0.9c 154 ± 29.8c

>20 7.4 ± 0.051a 0.071 ± 0.04d 7.89 ± 0.7d 2.6 ± 0.2d 4.1 ± 0.53d 0.28 ± 0.091d 181 ± 32.5d

Means in the same column followed by same letter do not differ significantly at P < 0.05, mean ± SEM. LU: land use, EX: exclosure, GL: Grazing land, US: upper slope, MS:middle slope, FS: foot slope.

Table 7Pearson's correlation coefficient between mycorrhizal structural colonization, spore density and soil properties.

HC MHC AC VC PH EC Av.P OM TN Av.K

SD 0.34 0.15 �0.18 �0.18 �0.44* �0.6** �0.27 �0.05 �0.00 �0.41HC 0.93** �0.45** �0.36 �0.36 �0.50* �0.3 0.14 0.08 �0.40MHC �0.32 �0.25 �0.15 �0.23 �0.26 0.34 0.25 �0.24AC 0.89** 0.60** 0.44* �0.21 �0.12 �0.04 �0.03VC 0.45** 0.29 �0.21 �0.05 0.14 �0.10PH 0.58** �0.13 0.05 �0.10 0.16EC 0.29 0.43* 0.28 0.45*

Av.P 0.11 0.24 0.85**

OM 0.65** 0.27TN 0.37

Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed).SD: spore density, HC: hyphal colonization, MHC: mycorrhiza hyphalcolonization, AC: arbuscular colonization, VC: vesicular colonization, EC: electrical conductivity (ms/m), Av.P: available phosphorus (ppm), OC: organic carbon (%), OM: organicmatter (5), TN: total nitrogen (%), Av.K: available potassium (ppm).

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e10 9

has no significant difference between exclosures and grazing lands.Short term grazing exclusion has no significant difference on soilpH between free grazing and grazing exclusion (Lu et al., 2015). Incontrary, soil pH was lower in non-grazed rangelands comparedwith grazed rangelands most likely because of the addition oflivestock urine, which increased soil pH greatly due to the hydro-lysis of urine in grazed grassland (Raiesi and Riahi, 2014).

Spore density and available phosphorus do not significantlydiffer (P > 0.05). Diversity and abundance of arbuscular mycorrhizalfungi associated with acacia trees from different land use systemsin Ethiopia showed negative correlation between the root coloni-zation levels and the available P concentration in soil (Zerihun et al.,2013). The reduction in spore density with an increase in soilavailable P can be attributed to the fact that, available soil phos-phorus suppresses AMF root colonization as well as AMF sporedensity (Galvez et al., 2001). Soil phosphorus did not show corre-lation with total root colonization in the present study. The lowphosphorus content of the soil could be ecologically significant forAMF in phosphorus uptake and the colonization potential is high insoils where the P concentration is low.

5. Conclusion

Exclosures improved the spore abundance andwoody plant rootcolonization of arbuscular mycorrhiza fungi of restored lands. Themiddle slope had more AMF within the exclosures. Age differencebrought a significant difference on AMF spore density, woody plantroot colonization and nutrient availability. Exclosures are impor-tant in restoring AMF in disturbed degraded areas. Age and slopeposition are key parameters to enumerate the AMF spore densityand root colonization to validate the restoration potential ofexclosures in the drylands. Quantifying the spore density and rootcolonization in restored areas using exclosures will increase the

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importance of exclosures as a potential indigenous AMF symbiont.

Acknowledgement

The Steps towards sustainable forest management with the localcommunities in Tigray, Northern Ethiopia (ETH 13/0018) projectfunded by NORAD under the NORHED programme fully fund thestudy. We are grateful to the two anonymous referees forconstructive comments on an earlier version of this manuscript.

References

Ajorlo, M., Abdullah, R., Hanif, A.H.M., Halim, R.A., Yusoff, M.K., 2011. Impacts oflivestock grazing on selected soil chemical properties in intensively managedpastures of Peninsular Malaysia. J. Trop. Agric. Sci. 34, 109e121.

An, G.O., Miyakawa, S., Kawahara, A., Osaki, M., Ezawa, T., 2008. Communitystructure of arbuscular mycorrhizal fungi associated with pioneer grass speciesMiscanthus sinensis in acid sulfate soils: habitat segregation along pH gradi-ents. J. Plant Nutr. Soil Sci. 54, 517e528. http://dx.doi.org/10.1111/j.1747-0765.2008.00267.x.

Bai, G., Bao, Y., Du, G., Qi, Y., 2013. Arbuscular mycorrhizal fungi associated withvegetation and soil parameters under rest grazing management in a desertsteppe ecosystem. Mycorrhiza 23, 289e301.

Baudron, F., Mamo, A.T., Dereje, A.M., 2015. Impact of farmland exclosure on theproductivity and sustainability of a mixed crop-livestock system in the CentralRift Valley of Ethiopia. Agric. Ecosyst. Environ. 207, 109e118. http://dx.doi.org/10.1016/j.agee.2015.04.003.

Birhane, E., Teketay, D., Barklund, P., 2007. Enclosures to enhance woody speciesdiversity in the dry lands of eastern Tigray, Ethiopia. East Afr. J. Sci. 1, 136e147.

Birhane, E., Kuyper, T.W., Sterck, F.J., Bongers, F., 2010. Arbuscular mycorrhizal as-sociations in Boswellia papyrifera (frankincense-tree) dominated dry deciduouswoodlands of Northern Ethiopia. For. Ecol. Manage. 260, 2160e2169. http://dx.doi.org/10.1016/j.foreco.2010.09.010.

Boddington, C.L., Dodd, J.C., 2000. The effect of agricultural practices on thedevelopment of indigenous arbuscular mycorrhizal fungi. II. Studies in experi-mental microcosms. Plant. Soil 218, 145e157. http://dx.doi.org/10.1023/A:1014911318284.

Borie, F., Rubio, R., Rouanet, J.L., Morales, A., Borie, G., Rojas, C., 2006. Effects oftillage systems on soil characteristics, glomalin and mycorrhizal propagules in aChilean Ultisol. Soil. Tillage. Res. 88, 253e261. http://dx.doi.org/10.1016/

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://

E. Birhane et al. / Journal of Arid Environments xxx (2017) 1e1010

j.still.2005.06.004.Bremmer, J.M., Mulvaney, C.S., 1982. Nitrogen total. In: Page, A.L. (Ed.), Methods of

Soil Analysis, Part 2 Chemical and Microbiological Properties ASA Monographnumber 9. Madison, WI, USA, 595e624.

Brundrett, M., 2004. Diversity and classification of mycorrhizal associations. Biol.Rev. 79, 473e495. http://dx.doi.org/10.1017/S1464793103006316.

Brundrett, M.C., Bougher, N., Dell, B., Grove, T., Malajczuk, N., 1996. Working withMycorrhizas in Forestry and Agriculture. ACIAR Monograph, vol. 32. AustralianCenter for International Agricultural Research, Canberra.

Carvalho, F., Souza, F.A., Carrenho, R., Moreira, F.M.S., Jesus, E.C., Fernandes, G.W.,2012. The mosaic of habitats in the high-altitude Brazilian rupestrian fields is ahotspot for arbuscular mycorrhizal fungi. Appl. Soil Ecol. 52, 9e19. http://dx.doi.org/10.1016/j.apsoil.2011.10.001.

Covacevich, F., Berbera, L., 2011. Indigenous Arbuscular mycorrhizae in areas withdifferent successional stages at a tropical dry forest biome in Brazile. Afr. J.Microbiol. Res. 5, 2697e2705. http://dx.doi.org/10.5897/AJMR11.435.

Dare, M.O., Abaidoo, R., Fagbola, O., Asiedu, R., 2013. Diversity of arbuscularmycorrhizal fungi in soils of yam (Dioscorea spp.) cropping systems in fouragroecologies of Nigeria. Arch. Agron. Soil Sc.i 59, 521e531. http://dx.doi.org/10.1080/03650340.2011.653682.

Dhillion, S.S., Gardsjord, T.L., 2004. Arbuscular mycorrhizas influence plant di-versity, productivity, and nutrients in boreal grasslands. Can. J. Bot. 82, 104e114.

Egbert, S.R.A., Mary, S.S., 2009. Studies on the status of arbuscular mycorrhizal fungion the fodder crop Sorghum bicolor (L.) moench. Trop. Life Sci. Res. 20, 99e109.

EMA, 2015. Ethiopian Metrological Agency (Addis Ababa, Ethiopia).Eriksson, A., 2001. Arbuscular mycorrhiza in relation to management history, soil

nutrients and plant species diversity. Plant Ecol. 155, 129e137. http://dx.doi.org/10.1023/A: 1013204803560.

Ferrol, N., Calvente, R., Cano, C., Barea, J.M., Aguilar, C.A., 2004. Analysing arbuscularmycorrhizal fungal diversity in shrub-associated resource islands from adesertification threatened semiarid Mediterranean ecosystem. Appl. Soil Ecol.25, 123e133. http://dx.doi.org/10.1016/j.apsoil.2003.08.006.

Galvez, L., Douds Jr., D.D., Drinkwater, L.E., Wagoner, P., 2001. Effect of tillage andfarming system upon VAM fungus populations and mycorrhizas and nutrientuptake of maize. Plant Soil 228, 299e308. http://dx.doi.org/10.1023/A:1004810116854.

Gee, G.W., Bauder, J.W., 1982. Particle size analysis. In: Klute, A. (Ed.), Methods ofSoil Analysis, Part 1 Physical and Mineralogical Methods. ASA Monographnumber 9. Madison, WI, USA, 383e411.

Habiyaremye, D., Cthrine, M., Viviene, M., John, N., Athnase, M., Vicky, R.,Motohito, Y., Fergus, S., 2015. Occurrence and abundance of arbuscularmycorrhizal fungi (AMF) in agro forestry systems of Rubavu and BugeseraDistricts in Rwanda. Afr. J. Microbiol. Res. 9, 838e846..

Hailemariam, M., Birhane, E., Zebene, A., Solomon, Z., 2013. Arbuscular mycorrhizalassociation of indigenous agroforestry tree species and their infective potentialwith Maize in the rift valley, Ethiopia. Agrofor. Syst. 87, 1261e1272. http://dx.doi.org/10.1007/s10457-013-9634-9.

Husband, R., Herre, E.A., Young, J.P.W., 2002. Temporal variation in the arbuscularmycorrhizal communities colonising seedlings in a tropical forest. FEMSMicrobiol. Ecol. 42, 131e136.

Inga, H., Sue, E., Sileshi, N., 2003. Flora of Ethiopia and Eritrea, vol. 4. Part 1 Apiaceaeto Dipsacaceae. Addis Ababa, Ethiopia.

Jeffries, P., Gianinazzi, S., Perotto, S., Turnau, K., Barea, J.M., 2003. The contributionof arbuscular mycorrhizal fungi in sustainable maintenance of plant health andsoil fertility. Biol. Fertil. Soils 37, 1e16. http://dx.doi.org/10.1007/s00374-002-0546-5.

Lakshman, H.C., Rajanna, L., Inchal, R.F., Mulla, F.I., Srinivasulu, Y., 2001. Survey ofVA- mycorrhizae in agroforestry and its implications on forest trees. Trop. Ecol.42 (2), 283e286.

Li, L.F., Zhang, Y., Zhao, Z.W., 2007. Arbuscular mycorrhizal colonization and sporedensity across different land-use types in a hot and arid ecosystem, SouthwestChina. J. Plant Nutr. Soil Sci. 170, 419e425. http://dx.doi.org/10.1002/jpln.200625034.

Lu, X., Yan, Y., Sun, J., Zhang, X., Chen, Y., Wang, X., Cheng, G., 2015. Short-termgrazing exclusion has no impact on soil properties and nutrients of degradedalpine grassland in Tibet, China. Solid. Earth. 6, 1195e1205. http://dx.doi.org/10.5194/se-6-1195-2015.

Lugo, M.A., Ferrero, M., Menoyo, E., Est�evez, M.C., Si~neriz, F., Anton, A., 2008.Arbuscular mycorrhizal fungi and rhizospheric bacteria diversity along analtitudinal gradient in south American Puna Grassland. Microb. Ecol. 55,705e713. http://dx.doi.org/10.1007/s00248-007-9313-3.

Mekuria, W., Aynekulu, E., 2013. Exclosure land management for restoration of thesoils in degraded communal grazing lands in northern Ethiopia. Land Degrad.Dev. 24, 528e538. http://dx.doi.org/10.1002/ldr.1146.

Muthukumar, T., Sha, L.Q., Yang, X.D., Cao, M., Tang, J.W., Zheng, Z., 2003. Distri-bution of roots and arbuscular mycorrhizal associations in tropical forest typesof Xishuangbanna, southwest China. Appl. Soil Ecol. 22, 241e253. http://dx.doi.org/10.1016/S0929-1393(02)00156-7.

Oehl, F., Sieverding, E., Ineichen, K., M€ader, P., Boller, T., Wiemken, A., 2003. Impactof land use intensity on the species diversity of arbuscular mycorrhizal fungi inagro ecosystems of central Europe. Appl. Environ. Microbiol. 69, 2816e2824.http://dx.doi.org/10.1128/AEM.69.5.2816-2824.2003.

Olsen, S.R., Sommers, L.E., 1982. Phosphorus. In: Page, A.L., et al. (Eds.), Methods of

Please cite this article in press as: Birhane, E., et al., Changes in arbuscularin response to exclosure age and slope position in the highlands of Tigdx.doi.org/10.1016/j.jaridenv.2017.03.002

Soil Analysis, Part 2: Chemical and Microbiological Properties. ASA Monograph9:403e430.

Pagano, M.C., Cabello, M.N., Scotti, M.R., 2010. Arbuscular mycorrhizal colonizationand growth of Eremanthus incanus Less. in a highland field. Plant Soil Environ.56, 412e418.

Picone, C., 2000. Diversity and abundance of arbuscular-mycorrhizal fungus sporesin tropical forest and pasture. Biotropica 32, 734e750. http://dx.doi.org/10.1111/j.1744-7429.2000.tb00522.x.

Pimentel, D., 2006. Soil erosion: a food and environmental threat. Environ. Dev.Sustain. 8, 119e137.

Raiesi, F., Riahi, M., 2014. The influence of grazing exclosure on soil C stocks anddynamics, and ecological indicators in upland arid and semi-arid rangelands.Ecol. Indic. 41, 145e154. http://dx.doi.org/10.1016/j.ecolind.2014.01.040.

Ranst Van, E., Verloo, M., Demeyer, A., Pauwels, J.M., 1999. Manual for the SoilChemistry and Fertility Laboratory: Analytical Methods for Soils and Plants,Equipments and Management of Consumables. University of Gent, Belgium.

Schnyder, H., Locher, F., Auerswald, K., 2010. Nutrient redistribution by grazingcattle drives patterns of topsoil N and P stocks in a low-input pastureecosystem. Nutrient Cycl. Agroecosyst. 88 (2), 183e195. http://dx.doi.org/10.1007/s10705-009-9334-z.

Seyoum, Y., Birhane, E., Mengistu, T., Esmael, N., Hagazi, N., Kassa, H., 2015.Enhancing the Role of the Forestry Sector in Building Climate Resilient GreenEconomy in Ethiopia: Scaling up Effective Forest Management Practices inTigray National Regional State with Emphasis on Area Exclosure. Center forinternational Forestry Research, Ethiopia Offics, Addis Ababa, Ethiopia.

Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis, third ed. Academic Press, NewYork.

Smith, S.E., Jakobsen, I., Grønlund, M., Smith, F.A., 2011. Roles of arbuscular my-corrhizas in plant phosphorus nutrition: interactions between pathways ofphosphorus uptake in arbuscular mycorrhizal roots have important implica-tions for understanding and manipulating plant phosphorus acquisition. PlantPhysiol. 156, 1050e1057. http://dx.doi.org/10.1104/pp.111.174581.

Songachan, L.S., Lyngdoh, I., Highland, K., 2011. Colonization of arbuscular mycor-rhizal fungi in moderately degraded sub-tropical forest stands of Meghalaya,Northeast India. J. Agric. Technol. 7, 1673e1684.

Tao, L., Jianping, L., Zhiwei, Z., 2004. Arbuscular mycorrhizas in a valley-typesavanna in southwest China. Mycorrhiza 14, 323e327. http://dx.doi.org/10.1007/s00572-003-0277-y.

Trejo, D., Barois, I., Sangabriel-Conde, W., 2016. Disturbance and land use effect onfunctional diversity of the arbuscular mycorrhizal fungi. Agrofor. Syst. 90,265e279. http://dx.doi.org/10.1007/s10457-015-9852-4.

Turrini, A., Sbrana, C., Strani, P., Pezzarossa, B., Risaliti, R., Giovannetti, M., 2010.Arbuscular mycorrhizal fungi of a mediterranean island (pianosa), within aUNESCO biosphere reserve. Biol. Fertil. Soils 46, 511e520. http://dx.doi.org/10.1007/s00374-010-0446-z.

van der Waal, C., Kool, A., Meijer, S.S., Kohi, E., Heitk€onig, I.M.A., de Boer, W.F., vanLangevelde, F., Grant, R.C., Peel, M.J., Slotow, R., de Knegt, H.J., Prins, H.H., deKroon, H., 2011. Large herbivores may alter vegetation structure of semi-aridsavannas through soil nutrient mediation. Oecologia 165, 1095e1107. http://dx.doi.org/10.1007/s00442-010-1899-3.

Verbruggen, E., Van Der Heijden, M.G., Weedon, J.T., Kowalchuk, G.A., R€oling, W.F.,2012. Community assembly, species richness and nestedness of arbuscularmycorrhizal fungi in agricultural soils. Mol. Ecol. 22, 1e13. http://dx.doi.org/10.1111/j.1365-294X.2012.05534.x.

Wang, Y.Y., Vestberg, M., Walker, C., Hurme, T., Zhang, X.P., Lindstro€m, K., 2008.Diversity and infectivity of arbuscular mycorrhizal fungi in agricultural soils ofthe Sichuan Province of mainland China. Mycorrhiza 18, 59e68. http://dx.doi.org/10.1007/s00572-008-0161-x.

Wiseman, P.E., Wells, C., 2005. Soil inoculum potential and arbuscular mycorrhizalcolonization of acer rubrum in forested and developed landscapes. J. Arboric. 31,296e302.

WRB, 2006. World Reference Base for Soil Resources, second ed. World Soil Re-sources Reports No. 103. FAO, Rome.

Wubet, T., Kottke, I., Teketay, D., Oberwinkler, F., 2003. Mycorrhizal status ofindigenous trees in dry Afromontane forests of Ethiopia. For. Ecol. Manag. 179,387e399. http://dx.doi.org/10.1016/S0378-1127(02)00546-7.

Yang, W., Zheng, Y., Gao, C., He, X., Ding, Q., Kim, Y., Rui, Y., Wang, S., Guo, L., 2013.The arbuscular mycorrhizal fungal community response to warming andgrazing differs between soil and roots on the qinghai-tibetan plateau. PLoS One8, 1e11. http://dx.doi.org/10.1371/journal.pone.0076447.

Yayneshet, T., Eik, L.O., Moe, S.R., 2009. The effects of exclosures in restoringdegraded semi-arid vegetation in communal grazing lands in northernEthiopia. J. Arid. Environ. 73, 542e549. http://dx.doi.org/10.1016/j.jaridenv.2008.12.002.

Zerihun, B., Mauritz, V., Fassil, A., 2013. Diversity and abundance of arbuscularmycorrhizal fungi associated with acacia trees from different land use systemsin Ethiopia. Afr. J. Microbiol. Res. 7 (48), 5503e5515. http://dx.doi.org/10.5897/AJMR2013.6115.

Zhao, Z.W., Xia, Y.M., Qin, X.Z., Li, X.W., Cheng, L.Z., Sha, T., Wang, G.H., 2001.Arbuscular mycorrhizal status of plants and the spore density of arbuscularmycorrhizal fungi in the tropical rain forest of Xishuangbanna, southwestChina. Mycorrhiza 11, 159e162. http://dx.doi.org/10.1007/s005720100117.

mycorrhiza fungi spore density and root colonization of woody plantsray, Northern Ethiopia, Journal of Arid Environments (2017), http://