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Glomalin-related soil protein and waterrelations in mycorrhizal citrus (Citrustangerina) during soil water deficitYing-Ning Zoua, A.K. Srivastavab, Qiang-Sheng Wua & Yong-MingHuanga

a College of Horticulture and Gardening, Yangtze University,Jingzhou, Hubei 434025, People’s Republic of Chinab National Research Centre for Citrus, Nagpur 440 010,Maharashtra, IndiaAccepted author version posted online: 20 Nov 2013.Publishedonline: 11 Dec 2013.

To cite this article: Ying-Ning Zou, A.K. Srivastava, Qiang-Sheng Wu & Yong-Ming Huang (2014)Glomalin-related soil protein and water relations in mycorrhizal citrus (Citrus tangerina)during soil water deficit, Archives of Agronomy and Soil Science, 60:8, 1103-1114, DOI:10.1080/03650340.2013.867950

To link to this article: http://dx.doi.org/10.1080/03650340.2013.867950

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WRITTEN ARTICLE

Glomalin-related soil protein and water relations in mycorrhizal citrus(Citrus tangerina) during soil water deficit

Ying-Ning Zoua, A.K. Srivastavab, Qiang-Sheng Wua* and Yong-Ming Huanga

aCollege of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, People’sRepublic of China; bNational Research Centre for Citrus, Nagpur 440 010, Maharashtra, India

(Received 15 July 2013; accepted 17 November 2013)

Glomalin-related soil protein (GRSP), a glycoprotein of arbuscular mycorrhizal fungi(AMF) secreted into soil, governs the aggregate stability, but the role of GRSP in soiland plant water is sparsely studied. The 24-week-old red tangerine (Citrus tangerina)inoculated with Glomus etunicatum and G. mosseae were subjected to a soil drying for12 days as soil water deficit (SWD). Length of SWD significantly reduced mycorrhizalcolonization, soil hyphal length, and leaf and soil water potential (Ψ), but increasedtotal GRSP (T-GRSP), easily extractable GRSP (EE-GRSP), and proportion of water-stable aggregates (WSAs) in >0.25 mm size, irrespective of AMF source. The AMF-inoculated seedlings showed significantly higher T-GRSP, EE-GRSP, and leaf/soil Ψthan non-AMF seedlings during SWD. A significantly positive correlation wasobserved for mycorrhizal colonization versus leaf or soil Ψ, and hyphal length versusleaf Ψ, suggesting that root intra- and extra-radical hyphae participated in watertransport. Interestingly, in GRSP fractions, only T-GRSP was significantly positivelycorrelated with 0.25–1 and >0.25 mm WSA and negatively with leaf and soil Ψ. Theseresults revealed a strong glue function of T-GRSP (not EE-GRSP and hyphae) to alterthe proportional distribution of WSA size, thereby aiding toward prevention of soilwater loss for improving soil–plant water relations.

Keywords: arbuscular mycorrhizas; drought; glomalin-related soil protein; mycorrhizalhyphae; water-stable aggregates

Introduction

Arbuscular mycorrhizal fungi (AMF) are an important soil-inhabited beneficial micro-organism belonging to phylum Glomeromycota, capable of establishing an obligatesymbiosis with ~80% of the land plants (Schüβler et al. 2001). The arbuscular mycor-rhizal (AM) symbiosis is reported to enhance the uptake of water and nutrients from soils,but also consumes ~20% plant-fixed carbohydrates toward growth acceleration (Parniske2008). Earlier studies confirmed improvements in water relation of host plants treatedwith the AMF (Augé 2001; Allen 2009; Smith et al. 2010) through different possiblemechanisms including: direct water absorption by extra-radical hyphae (Querejeta et al.2003; Allen 2009), indirect effect on nutrient acquisition capacity (Fitter 1988), stomatalregulation and photosynthesis enhancement (Augé et al. 2008), and greater osmoticadjustment and antioxidant protected systems (Wu & Xia 2006; Wu & Zou 2009), besidesregulation of some special genes encoding plasma membrane aquaporins, binding pro-teins, and various antioxidant enzymes (Porcel et al. 2006, 2007; Fan & Liu 2011).

*Corresponding author. Email: wuqiangsh@163.com

Archives of Agronomy and Soil Science, 2014Vol. 60, No. 8, 1103–1114, http://dx.doi.org/10.1080/03650340.2013.867950

© 2013 Taylor & Francis

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AMF can also secrete an abundant and persistent glycoprotein, namely, glomalin(Wright & Upadhyaya 1996). Glomalin in soil is defined as glomalin-related soil protein(GRSP), the role of which is largely related to improved soil structure (Rillig 2004; Spohn& Giani 2010) through a glue function to stabilize macroaggregates (>0.25 mm) (Gadkar& Rillig 2006). Such GRSP-induced aggregate stability is more conspicuous underdrought (Wu et al. 2008) than under salinity (Kohler et al. 2009). On the other hand,GRSP is reported to conserve the loss of water and nutrients from the soil exposed toabiotic stresses (Nichols 2008). In mycorrhizal biology, researches have predominantlyconcentrated on the roles of GRSP in regulating soil organic carbon pool and aggregatestability under open field conditions (Rillig 2004). Whereas limited studies have focusedon the role of GRSP and extra-radical mycelium forms of AMF on various soil processesvis-à-vis water relations.

In the background of multiple roles of AMF and GRSP in soil aggregate formationmediating water infiltration and retention (Oades 1984), the present study was carried outwith the objectives to study: (1) the comparative efficacy of Glomus mosseae andG. etunicatum on mycorrhizal colonization, hyphal length and production of GRSP, and(2) the roles of GRSP in aggregate stability and water relations during soil water deficit(SWD) using C. tangerina Tanaka as a test plant inoculated with AMF.

Materials and methods

Experimental setup

A total of 12 treatments (three mycorrhizal treatments, namely G. mosseae, G. etunicatum,and non-AMF and four levels of soil drying, namely 0, 4, 8, and 12 days) replicated threetimes were tested in a randomized factorial design using pots under greenhouseconditions.

Seeds of red tangerine (C. tangerina Tanaka) sterilized with 5% H2O2 were sown intothe 5400 cm3 plastic pots, each containing 2.8 kg autoclaved (0.11 MPa, 121°C, 2 h)substrate as a mixture of soil, vermiculite (<4 mm size, AnHong Minerals ProcessingFactory, Linshou, China), and sphagnum (Sale Department of Taifengyuan NativeProducts, Fusong, China) in 2:1:1 (volume basis). Vermiculite and sphagnum served assoil conditioners. The soil from a citrus orchard of Yangtze University campus wastaxonomically classified as Xanthi-Udic Ferralsols (FAO system). The potting substratemixture had pH 6.0, Bray-P 14.6 mg kg−1, and water holding capacity of 26.4%. Fifteengrams of AMF inoculant containing infected roots, spores, and external hyphae wereplaced in 5 cm below seeds at the time of sowing. All the seedlings were grown undergreenhouse conditions (day/night temperature: 24/18°C; relative humidity: 70–95%; andphotosynthetic photon flux density: 672–893 μmol m−2 s−1), without additional fertilizertreatment. Before treatment of SWD, the AM and non-AM seedlings were watered withdeionized water at 3-day interval to maintain soil moisture at field water capacity, basedon the daily evaporation rate.

At 23rd week, a soil hygrometer sensor (PST-55-30-SF, WESCOR Inc., Logan, UT,USA) was placed in the pot at a depth of 12 cm. One week later, all the 12 pots from eachtreatment were watered to field water capacity by weighing using the daily evaporationrate as basis. Thereafter, the seedlings were subjected to a continuous SWD by with-holding water in these pots for 12 days. Representative plant and soil samples wereconsequently taken from three pots per treatment at 0, 4, 8, and 12 days of soil drying asvarying levels of SWD.

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Sampling and variable analysis

Soil water potential (soil Ψ) was determined, when the soil sensor was linked to aPSΨPRO Water Potential System (WESCOR Inc.). Leaf water potential (leaf Ψ) waslikewise recorded using a PSΨPRO Water Potential System with a leaf hygrometer(L-51A-SF, WESCOR Inc.).

After the determination of soil and leaf Ψ, the seedlings were harvested and thesamples adhering on root systems were gently shaken, collected as rhizospheric soil,air-dried, ground, and sieved (4 mm) for the analysis of WSA and GRSP. Fresh soilsamples were used to determine hyphal length according to the procedure suggested byBethlenfalvay and Ames (1987). Simply, 2 g soil samples were extracted with 50 mL ofphosphate buffer solutions (pH 7.0). One-milliliter of supernatant was mixed with 0.5 mLof 0.05% trypan blue in lactophenol and the mixture was heated at 70°C in a water bathfor 20 min. Fifteen-microliter subsample of the mixture was placed in a glass slide andobserved for hyphal length under a compound microscope.

Fractions of GRSP including easily EE-GRSP and T-GRSP were determined follow-ing the method as described by Bedini et al. (2009). One-gram soil sample was extractedwith 8 mL 20 mM citrate (pH 7.0) for 30 min at 121°C and 0.11 MPa for EE-GRSP and8 mL 50 mM citrate (pH 8.0) for 60 min at 121°C and 0.11 MPa for T-GRSP. Theextracted samples were centrifuged at 10,000 × g for 5 min, and the supernatants wereanalyzed by Bradford (1976) assay using bovine serum albumin as a standard.

Soil water-stable aggregates (WSAs) (0.25–4 mm size fractions) were analyzed usingwet-sieving method with a nest of sieves (1 and 0.25 mm) (Yan 1988).

Root AM colonization was determined after clearing the 1.0-cm root segments with10% KOH for 90 min at 90°C and staining with 0.05% trypan blue in lactophenol for5 min (Phillips & Hayman 1970).

Statistical analysis

Data were subjected to analysis of variance (ANOVA) with mycorrhizal treatments, day ofSWD, and interaction between mycorrhizal treatment and day of SWD as sources ofvariation, followed by the Fisher’s protected least significant difference (p < 0.01 and0.05). Correlation coefficients among different variables were calculated with Proc Corr inSAS (SAS Institute Inc. 2001).

Results and discussion

Mycorrhizal colonization, hyphal length, and leaf and soil Ψ

Short-term SWD induced a significant reduction in root mycorrhizal colonization from50.1% and 32.0% at 0 days to 4.5% and 1.0% at 12 days of SWD in the G. mosseae andG. etunicatum colonized seedlings, respectively (Table 1; Figure 1(a)). Likewise, hyphallength decreased from 61.6 and 25.9 cm g−1 to 13.1 and 10.1 cm g−1 during 0–12 days ofSWD in the G. mosseae and G. etunicatum colonized seedlings, respectively (Table 1;Figure 1(b)). In addition, G. mosseae-colonized seedlings showed significantly higher rootAM colonization and hyphal length in soil than G. etunicatum-colonized seedlings undervarying levels of SWD (Table 1; Figure 1), suggesting that G. mosseae had a betterpotential to tolerate such stress than G. etunicatum. Mycorrhizal colonization was sig-nificantly positively correlated with soil hyphal length (Table 2). Mycorrhizal fungi and

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SWD showed significant (p < 0.01) interaction effects on root mycorrhizal colonizationand soil hyphal length (Table 1).

Leaf and soil Ψ gradually decreased with the length of SWD as a function of soil drying(Table 1; Figure 2). Leaf Ψ was significantly higher in AMF-infected seedlings than in non-AMF seedlings during SWD (Figure 2(a)), and G. mosseae showed a proportionally higher

Table 1. Significance of the sources of variation for several indexes in mycorrhizal and non-mycorrhizal red tangerine seedlings exposed to soil water deficit.

Mycorrhizalcolonization

Hyphallength EE-GRSP T-GRSP

LeafΨ

SoilΨ

>1 mmWSA

0.25–1mmWSA

>0.25mmWSA

AMF ** ** ** ** ** ** ** ** **SWD ** ** ** ** ** ** ** ** **AMF × SWD ** ** NS ** * ** ** ** **

Notes: EE-GRSP, easily extractable glomalin-related soil protein; NS, not significant; SWD, soil water deficit;T-GRSP, total glomalin-related soil protein; WSA, water-stable aggregate; Ψ, water potential.*p < 0.05; **p < 0.01.

Myc

orrh

izal

col

oniz

atio

n (%

)

0

10

20

30

40

50

60

G. etunicatumG. mosseae

(a) a,x

b,x

c,x

d,x

a,y

b,y

c,y

d,y

Days of soil water deficit0 4 8 12

Hyp

hal l

engt

h (m

g–1

soi

l)

0.0

0.2

0.4

0.6

a,y

a,x

b,y

b,x

c,x c,xc,y

c,x

(b)

Figure 1. Mycorrhizal colonization (a) and hyphal length (b) of citrus (Citrus tangerina) seedlingscolonized by Glomus mosseae, and G. etunicatum during a soil drying episode, respectively. Data(mean ± SE, n = 3) followed by different letters above the bars between soil water deficit treatmentsunder a given AMF inoculation (a, b, c, d) or between AMF treatments under a given soil waterdeficit (x, y) are significantly different (p < 0.01).

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Table2.

Pearson

correlationcoefficientsam

ongeach

variable(n

=8betweenmycorrhizalcolonizatio

nor

hyph

alleng

thandothervariables;n=12

amon

geach

variable

except

mycorrhizal

colonizatio

nandhy

phal

leng

th).

Mycorrhizal

colonizatio

nHyp

halleng

thEE-G

RSP

T-GRSP

LeafΨ

SoilΨ

>1mm

WSA

0.25

–1mm

WSA

>0.25

mm

WSA

Mycorrhizal

colonizatio

n1.00

0.94

**−0.22

−0.82

*0.96

**0.79

*−0.03

−0.59

−0.68

Hyp

halleng

th1.00

−0.25

−0.64

0.82

*0.60

−0.13

−0.42

−0.56

EE-G

RSP

1.00

0.34

0.13

0.27

0.02

0.44

0.52

T-GRSP

1.00

−0.71

*−0.73

**−0.14

0.76

**0.78

**LeafΨ

1.00

0.90

**0.18

−0.51

−0.48

SoilΨ

1.00

0.08

−0.45

−0.46

>1mm

WSA

1.00

−0.48

0.03

0.25

–1mm

WSA

1.00

0.86

**>0.25

mm

WSA

1.00

Notes:EE-G

RSP,

easily

extractableglom

alin-related

soilprotein;

NS,notsignificant;SWD,soilwater

deficit;

T-GRSP,

totalglom

alin-related

soilprotein;

WSA,water-stable

aggregate;

Ψ,water

potential.

*p<0.05;**p<0.01.

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effect on leaf Ψ than G. etunicatum. Soil Ψ was likewise significantly higher in AMF thannon-AMF seedlings during 4–12 days of SWD (Figure 2(b)). Moreover, inoculation withG. mosseae exhibited higher soil Ψ than with G. etunicatum during 4–12 days of SWD. Asignificant interaction effect of AMF and SWD was observed on leaf and soil Ψ (Table 1).Mycorrhizal colonization was significantly positively correlated with leaf and soil Ψ, andsignificantly negatively correlated with T-GRSP (Table 2). The highly positive correlationbetween root mycorrhizal colonization and leaf or soil Ψ suggested that mycorrhizal hyphaeare involved in absorption of water from soils (Allen 2009), thereby, enhancing leaf Ψ of thehost plant. These observations strongly support the fact that water transport of extra-radicalhyphae is more important under SWD conditions than under saturated soils, since AMFhyphae can effectively penetrate into soil pores to absorb the inaccessible water, thereby,providing the AMF roots more access to available soil water (Sánchez-Díaz & Honrubia1994; Wu et al. 2013).

AMF and GRSP production

Our study showed a strong influence of AMF colonization on the production of GRSPunder SWD (Table 1; Figure 3). The concentration of T-GRSP was higher in G. mosseae-colonized (0.37–0.68 mg g−1) and G. etunicatum-colonized (0.36–0.58 mg g−1) seedlings

Wat

er p

oten

tial

(M

pa)

–4

–3

–2

–1

0

Days of soil water deficit0 4 8 12

–0.8

–0.6

–0.4

–0.2

0.0

Non-AMFG. etunicatumG. mosseae

(a) Leaf

(b) Soil

d,z

c,z

b,z

a,z

d,y

c,y

b,y

a,y

d,x

c,x

b,x

a,x

c,z

b,z

a,z

c,y

b,y

d,x

c,x

b,x

a,x

d,x d,x

a,y

Figure 2. Leaf water potential (a) and soil water potential (b) of citrus (Citrus tangerina) seedlingscolonized by Glomus mosseae, G. etunicatum, and non-AMF during a soil drying episode, respec-tively. Data (mean ± SE, n = 3) followed by different letters above the bars between soil waterdeficit treatments under a given AMF inoculation (a, b, c, d) or between AMF treatments under agiven soil water deficit (x, y, z) are significantly different (p < 0.01).

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than in non-AMF seedlings (0.28–0.36 mg g−1) during SWD (Figure 3(b)). Meanwhile,G. mosseae-infected seedlings recorded significantly higher concentration of T-GRSPthan G. etunicatum-infected counterpart at 8 and 12 days of SWD. In addition, AMFand SWD interacted significantly on T-GRSP concentration without influencing EE-GRSP (Table 1), implying that T-GRSP is the metabolically more active fraction ofGRSP. Previous studies have shown that moderate SWD enhanced the GRSP concentra-tion, more in mycorrhizal than in non-mycorrhizal soils (Wu et al. 2008; Kohler et al.2009). Our study showed that length of SWD was directly proportional to the concentra-tion of T-GRSP (Figure 3(b)), while EE-GRSP increased up to 8 days of SWD (Figure 3(a)). The concentration of both T-GRSP and EE-GRSP in the rhizosphere of both G.mosseae- and G. etunicatum-infected seedlings was, hence, higher than of non-AMFseedlings (Figure 3). GRSP remains tightly bound in the fungal mycelia (Rillig &Mummey 2006) and related positively with extent of root AMF colonization and soilhyphae (Curaqueo et al. 2010; Wu et al. 2012). However, in the present study, onlyT-GRSP was significantly negatively correlated with substrate hyphal length (Table 2). Itappears that the SWD strongly mediated the release of GRSP by AMF hyphae. Reductionin mycorrhizal colonization and hyphal length induced during the SWD caused death/senescence of mycorrhizal hyphae, and the GRSPs bound in fungal hyphae as a hyphal

GR

SP f

ract

ion

conc

entr

atio

n (m

g g–1

soi

l)

0.0

0.2

0.4

0.6

0.8

a,xa,xa,xa,x

ab,xbc,x c,ya,yab,y bc,yc,y

a,x

Days of soil water deficit0 4 8 12

0.0

0.2

0.4

0.6

Non-AMFG. etunicatumG. mosseae

a,x

b,x

a,y

b,yc,x

d,x

a,z

b,zc,y

d,y

d,x

c,x

(a) EE-GRSP

(b) T-GRSP

Figure 3. Easily extractable glomalin-related soil protein (EE-GRSP) (a) and total glomalin-relatedsoil protein (T-GRSP) (b) concentration in the rhizosphere of citrus (Citrus tangerina) seedlingscolonized by Glomus mosseae, G. etunicatum, and non-AMF during a soil drying episode, respec-tively. Data (mean ± SE, n = 3) followed by different letters above the bars between soil waterdeficit treatments under a given AMF inoculation (a, b, c, d) or between AMF treatments under agiven soil water deficit (x, y, z) are significantly different (p < 0.01).

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wall component are, hence, released into the soil, resulting in the increase of GRSPfractions (Driver et al. 2005).

GRSP and WSAs

GRSP, an operationally defined soil carbon pool that is linked to AMF, with a slowturnover rate in soil, contributes lasting effects on soil aggregates (Rillig 2004). Glomalinis considered as microbial glue in aggregate formation, especially toward WSA of0.25 mm size (Gadkar & Rillig 2006; Wright et al. 2007). In addition, other factorssuch as hyphal network and root exudates are also involved in the WSA formation(Nichols 2008), since a positive correlation is commonly observed between GRSP andWSA stability (Rillig 2004).

Compared with non-AMF control, G. etunicatum-infected seedlings showed a higherproportion of >1 mm size WSA during the SWD (Table 1; Figure 4(a)), whileG. mosseae-infected seedlings indicated a significantly higher proportion of WSA in0.25–1 mm size (Table 1; Figure 4(b)). Higher proportion of >0.25 mm size WSA rankedas the order of G. mosseae-treated > G. etunicatum-treated > non-AMF-treated seedlingsduring the SWD (Table 1; Figure 4(c)). Herein, AMF, SWD, and the interaction of AMFand SWD significantly affected the proportion of >1, 0.25–1, and >0.25 mm WSA(Table 1). The inoculation with AMF facilitated the formation of soil aggregates, andtheir stabilization is viewed from the important consequences for soil carbon storagethrough physical protection of carbon inside of soil aggregates (Six et al. 2000).

Among GRSP fractions, only T-GRSP showed significant correlations with leaf andsoil Ψ, 0.25–1.00 mm WSA and >0.25 mm WSA, respectively (Table 2). Theseobservations further suggested that T-GRSP is more active than EE-GRSP. Earlier studiesshowed a significantly positive correlation between mycorrhizal colonization and T-GRSPin a citrus rhizosphere (Wang et al. 2011; Wu et al. 2012). It is well known that extra-radical mycelia networks can directly mediate in soil aggregate stability by meshing soilparticles together (Peng et al. 2013). However in the present study, there was nosignificant correlation between root AM colonization or hyphal length and percentageof WSA fractions in >1, 0.25–1.00, and >0.25 mm size, whereas T-GRSP but notEE-GRSP was significantly positively correlated with 0.25–1.00 mm and >0.25 mmWSA (Table 2). The results are in agreement with the findings of Rillig et al. (2002) inUltic Haploxeralf soil of Mendocino, CA, USA. These results revealed that T-GRSP butnot EE-GRSP was more actively involved in the WSA formation under the SWD, andT-GRSP much stronger mediated the WSA formation as compared with mycorrhizalfungal hyphae under the SWD. The lower soil hypahl length (0.10–0.62 m g−1) in ourstudy developed a weaker twining between soil different WSA fractions, which is liable tobe easily disrupted and is insufficient to stabilize WSAs. Studies in the past are somewhatdivided on the issue of type of relationship between GRSP and WSA. Some studiesobserved a significantly positive linear correlation between GRSP and WSA (Spohn &Giani 2010; Wu et al. 2012), while others reported a curvilinear relationship (Wright &Upadhyaya 1998; Harner et al. 2004). Singh (2012) proposed that the relationshipbetween GRSP and WSA is applied to the hierarchically structured soils, in which organicmatter is the main binding agent followed by CaCO3.

Irrespective of G. mosseae or G. etunicatum, the mycorrhizal soils showed significantlyhigher WSA at >0.25 mm size (Table 1; Figure 4(c)), but without any significant correlation ofWSA in >1, 0.25–1, and >0.25 mm size with either leaf or soil Ψ (Table 2). Our resultssuggested that water relations of plant and soil were not directly affected by GRSP-induced

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improvements in WSAs. Rillig et al. (2010) used an in vitro bioreactor system in which an AMfungusG. intraradiceswas grown in a soil devoid of detectable living microbes, and the resultsshowed that the mycelium of this fungus contributed toward the maintenance of WSAs andincreased soil water repellency, as measured by water drop penetration time.

Relationship between GRSP, mycorrhizal inoculation, and leaf and soil Ψ

Mycorrhizal hyphae trigger the movement of water from soil to host plants (Allen 2009).As much as 4% of water stored in the hyphal compartment has been observed to be

0

10

20

30

40

50

Non-AMFG. etunicatumG. mosseae

Pro

port

ion

of W

SA (

%)

0

10

20

30

40

Days of soil water deficit0 4 8 12

0

10

20

30

40

(a) >1 mm

(b) 0.25–1 mm

(c) >0.25 mm

a,x a,y

b,z

ab,xb,x a,y a,y

b,y

a,xab,x

b,x

c,x

a,yb,y

c,y

d,xy

a,xb,z

c,y

d,y

a,x

b,x

b,yb,y a,yb,z

c,x

a,x

a,x

a,xb,x

c,xb,z

a,x a,x a,x

Figure 4. Proportion of water-stable aggregate (WSA) fractions in >1 (a), 0.25–1 (b), and >0.25 mm(c) size in the rhizosphere of citrus (Citrus tangerina) seedlings colonized by Glomus mosseae,G. etunicatum, and non-AMF during a soil drying episode, respectively. Data (mean ± SE, n = 3)followed by different letters above the bars between soil water deficit treatments under a given AMFinoculation (a, b, c, d) or between AMF treatments under a given soil water deficit (x, y, z) aresignificantly different (p < 0.01).

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transferred to root compartment through mycorrhizal hyphae under drought-like condi-tions (Khalvati et al. 2005). Strong correlation of mycorrhizal colonization versus leaf orsoil Ψ and hyphal length versus leaf Ψ (Table 2) suggested that the root intra-radical orextra-radical hyphae collectively participated in water transport, thereby improving thewater relations of the host plant.

Only T-GRSP in GRSP fractions was significantly negatively correlated with soil andleaf Ψ in the present study (Table 2). No significant correlation of EE-GRSP with soil andleaf Ψ in the present work (Table 2) may be due to that EE-GRSP was recently producedby AMF and also relatively more labile (Koide & Peoples 2013). Nichols (2008) proposedthat GRSP acts as a coating on fungal hyphae to prevent loss of water, particularly undersoil drought conditions. In addition, GRSP seems to form a hydrophobic layer on theaggregate surface, thereby, reducing water loss within aggregates under drought (Nichols2008). T-GRSP in our studies showed improvement in WSA sizes of 0.25–1 mm and>0.25 mm during the SWD (Table 2). According to Augé (2004), mycorrhizal soils aswell structured soils displayed comparatively higher available water than poorly struc-tured non-mycorrhizal soils. These results suggested that GRSP as a glue agent could binddifferent soil aggregates by altering their distribution of aggregate sizes in WSA forma-tion, thus, enhancing the water relations of mycorrhizal soil and plants subjected to SWD.

Conclusion

SWD in terms of soil drying increased T-GRSP and EE-GRSP concentration and theproportion of >0.25 mm size WSA but significantly decreased mycorrhizal colonization,hyphal length, and leaf and soil Ψ. Correlation analysis further revealed that root coloni-zation and hyphal length were not significantly correlated with all WSA fractions, andonly T-GRSP in GRSP fractions was significantly positively correlated with 0.25–1 and>0.25 mm WSA, suggesting that T-GRSP contributed more toward the transformation ofWSAs into different sizes than EE-GRSP and mycorrhizal hyphae. Additionally, roothyphae and soil hyphae may be directly involved in water uptake from the soil, becauseroot mycorrhizal colonization was significantly positively correlated with soil and leaf Ψ,and hyphal length also positively with leaf Ψ. These observations could provide somemechanistic pathways toward the role of AMF in redressal of various abiotic stresses.

AcknowledgmentsThis study was supported by the Natural Science Foundation of Hubei Province (2012FFA001), theNational Natural Science Foundation of China (31372017; 31101513), the Key Project of ChineseMinistry of Education (211107), and the Science-Technology Research Project of Hubei ProvincialDepartment of Education, China (Q20111301).

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