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Pedobiologia 56 (2013) 161– 167

Contents lists available at SciVerse ScienceDirect

Pedobiologia - International Journal of Soil Biology

jo ur nal ho mepage: www.elsev ier .de /pedobi

Diversity of arbuscular mycorrhizal fungi in red tangerine (Citrus reticulataBlanco) rootstock rhizospheric soils from hillside citrus orchards

P. Wanga,b, B. Shua, Y. Wangb, D.J. Zhanga, J.F. Liua, R.X. Xiaa,∗

a Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, Hubei Province, Chinab Institute of Citrus Research, Zhejiang Academy of Agricultural Sciences, Taizhou 318020, Zhejiang Province, China

a r t i c l e i n f o

Article history:Received 18 July 2012Received in revised form 26 February 2013Accepted 9 March 2013

Keywords:Arbuscular mycorrhizal fungiColonizationSpore and hyphal length densityDiversityCitrus orchardEnvironmental factors

a b s t r a c t

Root colonization, abundance of spores and hyphae, as well as species diversity of arbuscular mycorrhizal(AM) fungi were analyzed in citrus orchards along an altitudinal gradient. The citrus trees were heavilycolonized (50.87–77.45%) by native AM fungi. In citrus orchards located at <600 m above sea level (asl), werecorded more extensive hyphal and arbuscular colonization, and higher spore and hyphal length density.AM fungal colonization, spore density, and hyphal length density were closely correlated with edaphicfactors such as available phosphorus, pH, and organic matter. A total of 18 AM fungal species belonging to3 different orders, Archaeosporales (1 species), Diversisporales (7 species) and Glomerales (10 species),were identified on the basis of spore morphological characteristics. In orchards located at higher altitudes(≥700 m asl), we observed a significant decrease in species richness and Shannon–Wiener index values.However, in all of the surveyed orchards, Glomus aggregatum, Funneliformis mosseae and Rhizophagusintraradices were the dominant species. Isolate frequency and relative abundance of AM fungi exhib-ited clearly distinct distribution patterns among taxonomic families. Canonical correspondence analysisrevealed that the AM fungal community structure was significantly influenced by environmental factors,especially altitude, pH, soil moisture, and available nitrogen. Our data indicated that environmental fac-tors are important in determining AM fungal root colonization, propagule numbers, and species diversityin citrus orchards.

© 2013 Elsevier GmbH. All rights reserved.

Introduction

Most terrestrial plants form symbiotic associations with arbus-cular mycorrhizal (AM) fungi. The symbiosis can benefit the hostplants in several ways, e.g., by enhancing uptake of mineral nutri-ents (especially phosphate), improving water status, protectingagainst microbial pathogens (Smith and Read 2008; Wehner et al.2010), and facilitating the formation and maintenance of soil struc-ture and dynamics (van der Heijden et al. 2006). Consequently, AMfungi (AMF) are perceived as critical components of various ecosys-tems and play a crucial role in the maintenance of agroecosystemstability and sustainable agricultural development. Essentially, AMfungal communities composed of different species may induce dif-ferent growth responses in plants, and therefore have a potentialrole to play in determining ecosystem variability and productivity(Moora et al. 2004). Thus, the functioning of AM symbiosis in thefield should be considered at the community level of AMF.

∗ Corresponding author at: Shizishan Street, No.1, Hongshan District, Wuhan430070, Hubei Province, China. Tel.: +86 27 87281637.

E-mail addresses: peter wang81@163.com (P. Wang),renxuexia@mail.hzau.edu.cn (R.X. Xia).

Citrus is one of the most economically important fruit cropsthroughout the world. Most citrus trees are cultivated in moun-tainous areas of Southern China and are frequently challenged witha wide variety of abiotic stresses, such as nutritional deficiency,drought, and extreme temperature. Shrestha et al. (1995) reportedthat citrus trees colonized by AMF had larger leaf areas and higherleaf phosphorus concentrations and exhibited more vigorous pho-tosynthesis than non-colonized trees. AM fungal colonization hasalso been shown to alleviate low-temperature stress and enhancedrought tolerance in citrus seedlings (Wu and Xia 2004; Wu 2011).Furthermore, in a pot-culture experiment, Wu et al. (2008) demon-strated that inoculation of citrus seedlings with AM fungi improvedthe soil structure by increasing soil water-stable aggregates. Cruzet al. (2002) studied the rhizosphere network establishment of AMfungal hyphae between citrus trees and other mycorrhizal plantsand reported that the citrus trees benefited when an adequate levelof nutrient exchange was maintained in the network. Enhancedgrowth of citrus trees is therefore relatively dependent on AMF.

Despite the importance of AMF for sustainable citrus produc-tion, studies on the diversity of AM fungi in citrus field soilsare relatively scarce, particularly in China. Studies conducted inCalifornia and Florida (Nemec et al. 1981), Eastern Spain (Camprubiand Calvet 1996), Rio Grande do Sul, Brazil (de Souza et al. 2002),

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162 P. Wang et al. / Pedobiologia 56 (2013) 161– 167

and Northern Thailand (Youpensuk et al. 2008) have demonstratedthat AMF found in citrus soils generally belonged to Glomus sensulato species. Red tangerine (Citrus reticulata Blanco) is an importantcitrus rootstock used in the southwest region of China. However,information on AMF in red tangerine rhizospheric soils from cit-rus orchards is limited. In the present study, we investigated AMfungal colonization of red tangerine roots, spore density, hyphallength density, and species diversity in orchards located at differ-ent altitudes in Southern China, and identified AM fungal speciesbased on the spore morphological characteristics to evaluate therelationship between AM fungal species distribution patterns andenvironmental factors.

Materials and methods

Study site and sampling

The study was conducted in citrus orchards located in ZiguiCounty (110◦18′–111◦0′ E, 30◦38′–31◦11′ N) in the Three GorgesRegion, Southern China. This area has a semi-tropical monsoonclimate with an annual mean temperature of 16.7 ◦C (maximum42.9 ◦C and minimum −9.0 ◦C), and an annual mean precipitation of1013.1 mm. However inconsistent rainfall frequently results in sea-sonal drought. The hypsography of the area is extremely variable,and citrus plantations are located mainly on slopes. The calcare-ous purple soil (Regosol in FAO Taxonomy) accounts for 78.7% ofthe total plantation area. Citrus trees are planted according to themountainous terrain. A total of 15 orchards, each planted with15–17-year-old citrus trees (Robertson naval orange grafted on C.reticulata Blanco) were selected as the study sites. The orchardswere located along an altitudinal gradient at 200, 400, 600, 700 and800 m above sea level (asl). To preserve slopes from soil erosion,consistent no-tillage and natural grass soil management practices(grass growing between citrus tree rows except in the region ofcitrus tree roots in terraced land, removal of competitive weeds,cutting to control grass height, mowing grass and mulching undercitrus trees during the drought season) were used in all of the sur-veyed orchards. At the start of orchard establishment, the soils hadbeen slightly improved by the removal of large gravel deposits.

Three orchards were sampled at each altitude using the five-point sampling method. At each sampled point, three healthy citrustrees with a similar growth pattern were selected. After remov-ing the ground cover within the drip line of the tree canopy inSeptember 2009, we gathered fine roots and rhizospheric soils of asingle citrus tree from 4 directions (east, west, south, and north)at a depth of 0–30 cm. Soil samples were mixed thoroughly, togive a single replicate from each sampling point in each orchard.A total of 75 soil samples were collected from 15 citrus orchards.To evaluate AM fungal spores and hyphae, ∼1 kg of soil was air-dried for 2 weeks, carefully ground by hand, passed through a2-mm mesh screen, and stored at 4 ◦C until analysis. Root samples(ϕ ≤ 1 mm) were taken to the laboratory, washed carefully with tapwater to remove soil, cut into 1-cm-long segments, and fixed informalin/acetic acid/ethanol solution until determination of rootmycorrhizal colonization.

Assessment of soil properties

Selected soil properties were analyzed using the methodsdescribed by Tan (1996): humid oxidation with K2CrO7 for soilorganic matter (OM); alkaline hydrolysis diffusion for availablenitrogen (AN); sodium bicarbonate soil test phosphorus methodfor available phosphorus (AP); and potentiometric method for soilpH. Gravimetric soil moisture (SM) was determined immediately

after taking a 5.00 g sub-sample from each soil sample. All soil sub-samples for SM test were dried overnight at 105 ± 2 ◦C.

Determination of AM fungal colonization and hyphal lengthdensity

Colonization of various AM fungal structures in citrus roots wasvisualized by staining according to Koske and Gemma (1989), andexamined under a compound-light microscope (Olympus-BH-2,Tokyo, Japan). The percentage of root length with AM hyphae (RLH),the percentage of root length of arbuscules (RLA), and the percent-age of root length with vesicles (RLV) were estimated using themagnified intersection method (McGonigle et al. 1990). Soil hyphallength density (HLD) was determined according to Bethlenfalvayand Ames (1987), with the aid of an ocular micrometer under acompound-light microscope.

Analysis of AM fungal spores

For each soil sample, spores were obtained from 40 g ofwell-mixed soil, using the wet sieving and sucrose gradi-ent centrifugation technique. The total number of spores wascounted under a stereoscopic microscope (Tech-XTS-30, Bei-jing, China). Thereafter, spores and sporocarps of similar sizeand colour were separated from the spore mixture, counted,and mounted onto slides, using polyvinyl alcohol-lactoglycerolwith and without Melzer’s reagent (Morton 1988). The sporesand sporocarps were subsequently identified using a compoundmicroscope at 100–400× magnification. AM fungi were identi-fied based on morphological characteristics, following currentdescriptions and identification manuals (Schenck and Pérez 1990;http://invam.caf.wvu.edu).

The AM fungal community structure was described using thefollowing ecological measures: spore density (SD), species rich-ness (SR), relative abundance (RA), isolation frequency (IF), andShannon–Wiener index (H′). These measures were calculatedas follows: SD = the number of AMF spores per 100 g of soil;SR = the number of identified AMF species per 40 g soil sub-sample;RA = spore numbers of a species (family)/the total number of identi-fied spore samples × 100%; IF = the number of soil samples where aspecies (family) occurred/the total number of soil samples × 100%;H′ = −�(Pi) ln(Pi), where Pi = ni/N, ni is the number of individualsof the ith species, and N is the total number of individuals in allspecies.

Statistical analysis

Data for AM colonization rates were transformed to arc sin(x)1/2

prior to analysis. Data were subjected to one-way analysis of vari-ance (ANOVA), and means were compared by least significantdifference (LSD) at the 0.05 level with SAS 9.1 software. Correla-tion coefficients between AM symbiosis indices (with respect toAM colonization, SD, and HLD) and soil properties were consideredsignificant at P < 0.05 and highly significant at P < 0.01, respec-tively. The relationships among AM fungal distribution pattern andselected environmental variables, including altitude (ALT), soil OM,AN, AP, pH, and SM, were analyzed by canonical correspondenceanalysis (CCA) using CANOCO 4.5 software (ter Braak and Smilauer2002).

Results

Soil properties

In the present study, the highest OM (22.91 g kg−1) was mea-sured in orchards at 400 m asl, the highest AN (136.72 mg kg−1) and

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Table 1Soil characteristics in citrus orchards along an altitudinal gradient.

Altitude (m asl) Location OM (g kg−1) AN (mg kg−1) AP (mg kg−1) SM (%) pH value

200 30◦52′27.6′′ N 20.18 ± 0.49b 126.97 ± 1.92bc 12.96 ± 0.22c 16.01 ± 0.17d 5.94 ± 0.14a110◦44′29.0′′ E

400 30◦55′38.9′′ N 22.91 ± 0.61a 130.65 ± 1.72b 14.77 ± 0.39b 17.88 ± 0.27c 5.89 ± 0.12a110◦44′34.7′′ E

600 30◦55′5.6′′ N 21.18 ± 0.73ab 136.72 ± 1.67a 16.50 ± 0.53a 19.76 ± 0.69b 5.87 ± 0.14a110◦45′35.8′′ E

700 30◦56′7.6′′ N 19.97 ± 0.92 b 124.93 ± 1.17c 10.92 ± 0.41d 23.54 ± 0.25a 5.78 ± 0.14a110◦42′35.0′′ E

800 30◦54′37.6′′ N 16.60 ± 0.65c 122.25 ± 1.40c 6.50 ± 0.44e 24.50 ± 0.53a 5.56 ± 0.06b110◦46′25.0′′ E

Values in each column followed by different letters are significantly different (P < 0.05).

AP (16.50 mg kg−1) in orchards at 600 m asl, the highest pH (5.94)in orchards at 200 m asl, and the highest SM (24.50%) in orchards at800 m asl. The lowest OM (16.60 g kg−1), AN (122.25 mg kg−1), AP(6.50 mg kg−1) and pH (5.56) were measured in orchards at 800 masl, and the lowest SM (16.01%) in orchards at 200 m asl (Table 1).Our results showed a general trend, namely that higher soil fertil-ity was preserved at lower altitudes, while SM increased at higheraltitudes. In addition, AP was relatively low (<20 mg kg−1) in all ofthe surveyed citrus orchards.

Arbuscular mycorrhizal colonization, spore density, and hyphallength density

All of the citrus trees surveyed were colonized by indigenousAMF, and formed typical AM root structures such as intra- or inter-cellular hyphae, arbuscules, and vesicles (alone or in combination).Intra- or intercellular hyphae and arbuscules were abundant inmost citrus roots; however, vesicles and intraradical spores wereobserved only occasionally.

RLH and RLA showed a decreasing trend with increasing alti-tude. The highest values (77.45% and 51.59%, respectively) wererecorded in orchards at 200 m asl, the lowest values (50.87% and27.38%, respectively) in orchards at 800 m asl. By contrast, RLVshowed an increasing trend with increasing altitude. The highestvalue (3.73%) was observed in orchards at 800 m asl, and the lowestvalue (1.85%) in orchards at 200 m asl. ANOVA analysis revealed sig-nificant fluctuations in RLH, RLA, and RLV among orchards locatedat different altitudes (Fig. 1A). Additionally, RLH and RLA were pos-itively correlated with AP and pH, but negatively correlated withSM and altitude (Table 2).

In the present study, the highest SD (765.83 spores/100 g soil)was detected in orchards at 400 m asl, the highest HLD (2.57 m/gsoil) in orchards at 600 m asl, and the lowest SD and HLD (412.50spores/100 g soil and 1.39 m/g soil, respectively) in orchards at800 m asl (Fig. 1B). Similar to RLH and RLA, SD and HLD were notablyhigher in orchards located at lower altitudes (<600 m asl). In addi-tion, SD was positively correlated with OM, AN, and AP, and HLDwere positively correlated with OM, AN, AP, and pH (Table 2).

Diversity of AM fungi

A total of 18 AM fungal species, belonging to 3 differentorders, namely Archaeosporales, Diversisporales and Glomerales,were identified in all of the surveyed orchards. According to thephylum classification of Schübler and Walker (2010), 4 putativespecies were members of Acaulospora, 4 of Glomus, 2 each ofClaroideoglomus and Funneliformis, and 1 each of Archaeospora,Gigaspora, Pacispora, Scutellospora, Rhizophagus and Sclerocystis.Three species, Glomus aggregatum, Funneliformis mosseae andRhizophagus intraradices, were dominant in all of the surveyedorchards (Table 3).

Fig. 1. The different AM structure colonization rates (A) of citrus roots, spore densityand hyphal length density (B) in citrus orchards at different altitudes. Bars withthe same letter are not significantly different (P < 0.05), the same as below. Note:RLH: percentage of root length with AM hyphae, RLA: percentage of root length ofarbuscules, RLV: percentage of root length with vesicles, SD: spore density and HLD:hyphal length density.

The mean SR ranged from 7.33 in orchards at 800 m asl to 14.00in orchards at 400 m asl. The SR of orchards at lower altitudes wasmarkedly higher than that of orchards at higher altitudes (Fig. 2A).The Shannon–Wiener diversity index showed a similar decreasingtrend with increasing altitude; H′ varied from 1.22 in orchards at800 m asl to 1.95 in orchards at 400 m asl. H′ did not differ signifi-cantly among orchards located at <600 m asl, but decreased notablyin orchards located at 700 m asl and 800 m asl (Fig. 2B).

At the family level, the IF and RA of some AMF varied markedlyamong orchards located at different altitudes. The highest IF andRA were determined for Glomeraceae, followed by Claroideoglom-eraceae and Acaulosporaceae (Fig. 3). The IF of Glomeraceae was100% for all of surveyed orchards. The IF of Claroideoglomeraceaeremained at 100% in orchards located at <600 m asl, then declinedwith increasing altitude. No clear IF pattern for Acaulosporaceaeaccording to altitude was apparent (Fig. 3A). With regard to RA,Glomeraceae exhibited a clear increasing trend with increasingaltitude, and occurred with the highest abundance in orchards at

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164 P. Wang et al. / Pedobiologia 56 (2013) 161– 167

Table 2Correlation analysis between environmental factors and AM symbiosis indices in citrus orchards.

OM AN AP SM pH ALT

Hyphae colonization 0.630 0.469 0.722* −0.949** 0.934** −0.954**

Arbuscule colonization 0.590 0.408 0.675* −0.915** 0.912** −0.919**

Vesicule colonization −0.655* −0.406 −0.705* 0.905** −0.949** 0.932**

Spore density 0.689* 0.669* 0.746* −0.590 0.590 −0.418Hyphal length density 0.687* 0.881** 0.885** −0.635 0.692* −0.442

* Significant at P < 0.05.** Highly significant at P < 0.01.

Fig. 2. AM fungal species richness (A) and Shannon–Weiner index (B) in citrusorchards at different altitudes.

800 m asl. Claroideoglomeraceae was significantly more abundantin orchards located at <600 m asl than in orchards at 700 m asland 800 m asl. Acaulosporaceae was most numerous in orchardsat 400 m asl, but showed no notable differences in abundancebetween orchards located at lower and higher altitudes (Fig. 3B).

Influence of environmental variables on AM fungal sporecommunity

CCA revealed clear relationships between environmental vari-ables and AM fungal species distribution pattern. A total 79.1%cumulative variance of species and environment relation could beexplained by the first 2 ordination axes (axis 1, 57.3% and axis2, 21.8%; Fig. 4). The significance (based on the Monte Carlo per-mutation tests) of all canonical axes was P = 0.002, indicating thatenvironmental factors significantly influenced AM fungal sporecommunity structures in the surveyed citrus orchards.

In the Fig. 4 biplots, the angle between a vector and any axisindicates the correlation degree of the variable with the axis, whilethe length of a vector indicates the relative significance of the cor-relation of the variable with the axis. Accordingly, ALT, SM and pHwere correlated with the first axis resulting in the highest differ-ences in AM fungal species distribution. The Monte Carlo test forthis axis revealed a significance of P = 0.002. The second axis showed

Fig. 3. Isolate frequency (A) and relative abundance (B) of some AM fungal familiesin citrus orchards.

a significantly positive correlation with AN. In comparison withALT, SM, pH and AN, OM and AP exhibited a lesser degree of corre-lations with the first and second axes indicating that ALT, SM, pHand AN played the dominant role in determining the distributionof AM fungal species in the surveyed citrus orchards. Furthermore,in Fig. 4, AM fungal species triangles in proximity to each othercorrespond to species frequently occurring together. Accordingly,Acaulospora bireticulata, Acaulospora rehmii, Claroideoglomus etu-nicatum, Funneliformis caledonium, F. mosseae, G. aggregatum andR. intraradices showed similar distributions with regard to relativespecies abundance across the samples. These species were gener-ally isolated from citrus orchards with lower values of soil fertility,SM, pH and ALT.

Discussion

In the present study, we observed heavy AM colonization ofcitrus trees. This may have been caused by the relatively lowAP level in all of the surveyed citrus orchards. AM are recog-nized as key symbiotic microorganisms for the majority of plants,especially under conditions of P-limitation (Jeffries et al. 2003).A decreasing trend of hyphal and arbuscular colonization withincreasing altitude was observed. This finding is in accordancewith those of Wu et al. (2004) and Gai et al. (2012), who reported

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P. Wang et al. / Pedobiologia 56 (2013) 161– 167 165

Table 3Species list of AMF identified in citrus orchards at different altitudes.

AMF species Altitude (m asl)

200 400 600 700 800

a3(15) a3(18) a3(14) a2(12) a2(11)

ArchaeosporalesArchaeospora

Archaeospora schenckii(Sieverd. & S. Toro)Walker & A. Schüssler

+ + +

DiversisporalesAcaulospora

Acaulospora bireticulataF.M. Rothwell & Trappe

+ + + + +

Acaulospora rehmiiSieverd. & S. Toro

+ + + + +

Acaulospora spinosa C.Walker & Trappe

+ +

Acaulospora sp1 +Gigaspora

Gigaspora albida N.C.Schenck & G.S. Sm.

+ + + + +

PacisporaPacispora

chimonobambusae (C.G.Wu & Y.S. Liu) Sieverd. &Oehl ex C. Walker,Vestberg & A. Schüssler

+ + + + +

ScutellosporaScutellospora sp1 + + + + +

GlomeralesClaroideoglomus

Claroideoglomusclaroideum (N.C. Schenck& G. S. Sm.) C. Walker &A. Schüssler

+ + + + +

Claroideoglomusetunicatum (W.N. Becker& Gerd.) C. Walker & A.Schüssler

+ + + + +

FunneliformisFunneliformis

caledonium (T.H.Nicolson & Gerd.) C.Walker & A. Schüssler

+ +

Funneliformis mosseae(T.H. Nicolson & Gerd.) C.Walker & A. Schüssler

+b +b +b +b +b

GlomusGlomus aggregatum

N.C. Schenck & G.S. Sm.+b +b +b +b +b

Glomus tortuosum N.C.Schenck & G.S. Sm.

+ + + +

Glomus sp1 + + + +Glomus sp2 +

RhizophagusRhizophagus

intraradices (N.C.Schenck & G.S. Sm.) C.Walker & A. Schüssler

+b +b +b +b +b

SclerocystisSclerocystis clavispora

Trappe+ + +

a Order number (species number).b Dominant species of specific altitude.

that AM establishment was more difficult at higher altitudes. Inorchards located at higher elevations (700 m asl and 800 m asl), cit-rus trees are frequently challenged by extreme cold temperaturesin winter and early spring. This may suppress the developmentof AM symbiosis, because cold termperatures can constrain theamount of photosynthesized carbon that is shared with AMF athigher elevations (Ruotsalainen et al. 2002). On the other hand, wealso observed an increasing trend of vesicular colonization with

Fig. 4. Ordination diagram from the CCA of the relationship between the distri-bution of AMF species and environmental variables in citrus orchards along analtitudinal gradient. Note: The environmental variables were represented by vec-tors, the AM fungal species by empty triangles, where A. b: Acaulospora bireticulata,A. r: Acaulospora rehmii, A. s: Acaulospora spinosa, Ar. S: Archaeospora schenckii, C.c: Claroideoglomus claroideum, C. e: Claroideoglomus etunicatum, F. c: Funneliformiscaledonium, F. m: Funneliformis mosseae, G. a: Glomus aggregatum, G. t: Glomus tor-tuosum, G. sp1: Glomus sp1, Gi. a: Gigaspora albida, P. c: Pacispora chimonobambusae,R. i: Rhizophagus intraradices, S. c: Sclerocystis clavispora, Sc. sp1: Scutellospora sp1.

increasing altitude. This finding may be explained by the idea thatmore vesicles (storage structures formed by AMF) could functionas propagules in a rugged environment (Biermann and Linderman1983).

Well-developed AM colonization of roots enhances transporta-tion of organic nutrients from hosts to AMF, thereby improvinghyphal extension and sporulation. Accordingly, in the presentstudy, a higher density of AM fungal spores and hyphae wasdetected in orchards located at lower altitudes. Previous studieshave revealed that soil phosphatase activity is enhanced by anincrease in AM colonization, and that AMF may produce extracel-lular phosphatase (Joner et al. 2000; Chethan Kumar et al. 2008;Wang et al. 2011), thereby promoting mineralization of organicphosphorus for host plant uptake. In our study, soil AP was pos-itively correlated to SD, HLD, RLH and RLA. This was probably dueto an increase in soil phosphatase activity, mediated by AM symbio-sis in the rhizosphere. Hinsinger et al. (2003) reported that plantssurviving in P-deficient conditions could induce roots to excretelarge amounts of organic acids that would mobilize ineffective Psources; however, this was associated with rhizosphere acidifica-tion. We found that soil pH was positively correlated with RLH, RLAand HLD. Thus, it may be feasible to utilize AM symbiosis to inhibit adecrease in soil pH, while enhancing P uptake in the citrus orchardthat were studied.

Rillig et al. (2001) suggested that a common hyphal networkof AMF and the concomitant secretion of glomain could repre-sent an important source of soil organic carbon and nitrogen.This concept is supported by the results of our present study, inwhich soil OM and AN were positively correlated with HLD andSD. Additionally, the hyphae of AMF provided an increased areafor interactions with other plant growth-promoting rhizobacteria,and therefore represent an important pathway for the transloca-tion of energy-rich plant assimilates to the soil (Johansson et al.2004).

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We identified a total of 18 AM fungal species, based on sporemorphological characters. This number is comparable with thosepreviously recorded in other citrus orchards but much lower thanthose occurring in the majority of natural ecosystems (Oehl et al.2003; Zhao and Zhao 2007; Tian et al. 2009; Gai et al. 2012).Menéndez et al. (2001) reported that, in comparison with natu-ral ecosystems, agricultural soils generally showed low AM fungaldiversity. This is probably attributed to the use of conventional agri-cultural practices (e.g., fertilizers, pesticides, herbicides, and tillage)which reduced the AMF in agricultural soils, particularly in terms ofnumbers of species (Helgason et al. 1998). Additionally, the long-term monoculture in orchard systems may select for AM fungalspecies that provide limited benefits to citrus plants (Gosling et al.2006).

The most common AMF in citrus rhizospheric soils were Rhi-zophagus fasciculatus in California, F. mosseae and R. intraradices inEastern Spain, Glomus macrocarpum in Rio Grande do Sul, Brazil,C. etunicatum and Acaulospora scrobiculata in Northern Thailand(Nemec et al. 1981; Camprubi and Calvet 1996; de Souza et al. 2002;Youpensuk et al., 2008), most of which broadly fall into the Glomer-aceae family. The dominant AM fungal species (i.e., G. aggregatum,F. mosseae and R. intraradices) identified in the present study, arealso members of Glomeraceae family. Oehl et al. (2003) previouslyreported that AM fungal species belonging to Glomeraceae familyoften dominate in agricultural soils. In our investigation, we furtherobserved that the same 3 species were dominant in all of the sur-veyed orchards, possibly because of the limited range of altitudessampled and the uniform natural grass-soil management practiceused.

Despite the lack of variation in dominant AM fungal species,SR and H′ differed notably between orchards located at lower andhigher altitudes. Lower SR and H′ were recorded at higher (700 masl and 800 m asl) altitudes. These findings for a cultivated ecosys-tem are in accordance with the generally observed pattern of SRand H′ in natural ecosystems. For instance studies of the Andesand Rocky Mountains (Schmidt et al. 2008), the South AmericanPuna grasslands (Lugo et al. 2008), and sloped of the Segrila Moun-tain (Gai et al. 2012) have revealed decreasing trends of SR and H′

with increasing elevation. Moreover, apart from AM fungal SR andH′, we demonstrated that the spore RA and IF of Glomeraceae andClaroideoglomeraceae varied markedly among orchards located atdifferent altitudes.

Soil properties are heavily affected by altitudinal gradients(Ruotsalainen et al. 2009; Djukic et al. 2010), and this may affectthe distribution of AM fungal species (Hayman 1982). In fact, weobserved a marked variation in soil factors such as SM and ANbetween orchards located at different altitudes; moreover, the CCArevealed that these variations significantly influenced the AM fun-gal species community structure. Although no notable variations insoil pH were recorded between the different altitudes in our study,the distribution of AM fungal species is known to be extremelysensitive to soil pH (Tian et al. 2009).

In conclusion, this study clearly demonstrated that AM fungalroot colonization, hyphal extension and sporulation, as well as fun-gal species diversity showed clear decreasing trends in orchardslocated at higher altitudes. Moreover, we have revealed the AMfungal species distribution pattern is significantly influenced byenvironmental variables in citrus orchards. Environmental factorssuch as altitude, SM, pH, and AN significantly influenced AM fungalspecies community structure. Therefore, in order to maximize thebenefits of AMF in sustainable citrus production, we propose thatorganic agricultural practice be employed to improve the microe-cology of citrus orchards. Such practices have previously beenshown to increase AM fungal root colonization, propagule num-bers and species diversity (Oehl et al. 2004). It should be notedthat the identification of AM fungal species in our study was based

on the traditional spore morphological method. This probably didnot accurately reflect all AMF-colonizing citrus roots. Moreover, theAMF species that bore few or no spores were often easily neglectedin field investigations. Additionally, some of the AMF what we iden-tified may be associated with grass roots instead of the plantedcitrus trees given the limitations of the sampling strategies in cit-rus orchards. Thus, further studies are required to more fully andprecisely understand the molecular ecology of symbiotic associ-ation formed between AMF and citrus trees by using molecularidentification approaches.

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