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Soil Biology & Biochemistry 45 (2012) 181e183

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Soil Biology & Biochemistry

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Short communication

Spatial distribution of glomalin-related soil protein and its relationshipswith root mycorrhization, soil aggregates, carbohydrates, activityof protease and b-glucosidase in the rhizosphere of Citrus unshiu

Qiang-Sheng Wu a,*, Xin-Hua He b,c,d, Ying-Ning Zou a, Kai-Ping He a, Ya-Hong Sun a, Ming-Qin Cao a

aCollege of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, Chinab State Centre of Excellence for Ecohydrology, Edith Cowan University, Joondalup, WA 6027, AustraliacCentre for Ecosystem Management/School of Natural Sciences, Edith Cowan University, Joondalup, WA 6027, Australiad School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia

a r t i c l e i n f o

Article history:Received 14 June 2011Received in revised form18 August 2011Accepted 9 October 2011Available online 20 October 2011

Keywords:b-glucosidaseCitrusGlomalinGlomalin-related soil proteinMycorrhizationProtease

* Corresponding author. Tel./fax: þ86 716 8066262E-mail address: wuqiangsh@163.com (Q.-S. Wu).

0038-0717/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.soilbio.2011.10.002

a b s t r a c t

Relationships between the spatial distributions of glomalin-related soil protein (GRSP) and soil aggre-gates, carbohydrates or relevant enzymes are poorly studied. We found that two categories of GRSP, theeasily extractable Bradford-reactive soil protein (EE-BRSP) and total BRSP (T-BRSP), respectively rangedbetween 0.3e0.6 and 0.5e0.8 mg/g DW soil, and these two BRSPs decreased with the increase of soildepth (0e40 cm) in the rhizosphere of a 22-year-old Citrus unshiu orchard. Both EE-BRSP and T-BRSPwere significantly positively correlated with mycorrhization, 0.25e0.50 mm soil water-stable aggregates,water-extractable or hydrolyzable carbohydrates, and b-glucosidase, but significantly negatively corre-lated with protease. Our results demonstrate that the spatial distribution of GRSP is significantly affectedby mycorrhization, soil carbohydrate, b-glucosidase and protease.

� 2011 Elsevier Ltd. All rights reserved.

As an insoluble N-linked glycoproteinwithw37% carbon (C) and3e5% nitrogen (N), glomalin contributes to soil structure stabiliza-tion andC pools (Wright andUpadhyaya,1998; Lovelock et al., 2004).Glomalin is mainly produced by arbuscular mycorrhizal fungi (AMF)hyphae and quantified from soils as glomalin-related soil protein(GRSP). The GRSP is often related to soil C pool (Treseder and Turner,2007), though glomalin has limited contribution to C budgets inshort-term experiments (Driver et al., 2005). Less information isavailable to the spatial distributions of GRSP in the soil profile and itsrelationships with root and soil carbohydrates, though glomalin isused to evaluate soil quality (He et al., 2010).

Soil b-glucosidase is a useful soil quality indicator related to Ccycling (Stott et al., 2010), whereas soil protease is highly correlatedwith soil N transformation (Rejsek et al., 2008). However, it isunclear if soil b-glucosidase or protease is related to GRSP, whichcontains both C and N.

Citrus strongly depends on AMF in the field (Wu and Xia, 2006).To better understand the relationships between the formation of

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GRSP and relevant soil variables, we first assessed the spatialdistribution of GRSP, carbohydrates, water-stable aggregates(WSA), b-glucosidase and protease in the rhizosphere of Citrusunshiu (cv. Guoqing-1 grafted on Poncirus trifoliata), and thencorrelated GRSP with these variables.

The experiment was conducted in a campus orchard thathas w400 22-year-old citrus trees belonging to Yangtze University,China. Sixteen similar plants were randomly selected from fourblocks (4-trees� 4-blocks). Soils (Xanthi-udic ferralsol) and rootswere collected at 0e10, 10e20, 20e30 and 30e40 cm depth withina 2 m radius of tree canopy on 26 May 2010. Samples from 4-trees/block were mixed as one composite sample. Part of fresh sampleswas for mycorrhization and enzyme analysis. The rest was air-driedand ground (4 mm) for other analysis.

The GRSP is assayed by the Bradford method as Bradford-reactive soil proteins (BRSPs), which include the easily extractableBRSP (EE-BRSP) and total BRSP (T-BRSP) (Rosier et al., 2006).Determination of EE-BRSP and T-BRSP was carried out followingBedini et al. (2009), WSA following Yan (1988), soluble sugarfollowing Yemm and Willis (1954), soil hot water-extractable andhydrolyzable carbohydrates following Li et al. (2002), b-glucosidaseactivity following Stott et al. (2010), soil protease following

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Fig. 1. Spatial distribution of root mycorrhizal colonization (%, open bars) and rootsoluble sugar concentration (mg/g DW, solid bars) in the rhizosphere of 22-year-oldCitrus unshiu. Data (means� SE, n¼ 4) followed by different letters (a, b, c) above thebars are significantly different among soil depths for each variable at P< 0.05.

Q.-S. Wu et al. / Soil Biology & Biochemistry 45 (2012) 181e183182

Cao et al. (1982), and AM colonization following Phillips andHayman (1970).

Significantly higher root AM mycorrhization of C. unshiu rankedat 0e10>10e20z 20e30> 30e40 cm soil depth (Fig. 1), which isconsistent with results from C. aurantium and P. trifoliata� C. limon(Levy et al., 1983). Root AM colonization was significantly nega-tively correlated with 0.5e1.0 mm size soil WSA and protease(r¼�0.57e0.93, P< 0.05), but significantly positively correlatedwith root soluble sugar, soil hot water-extractable and hydrolyzablecarbohydrates, EE-BRSP, T-BRSP, and soil b-glucosidase(r¼ 0.67e0.94, P< 0.01).

Both EE-BRSP and T-BRSP were highest at 0e10 and 10e20 cmand higher at 20e30 cm than at 30e40 cm soil depth (Table 1), andEE-BRSP was significantly positively correlated with T-BRSP(r¼ 0.89, P< 0.01). A range of 0.3e0.6 mg EE-BRSP/g soil in therhizosphere of C. unshiu (Table 1) is consistent with those(0.3e0.6 mg/g) found in theMuUs sandland, China (Bai et al., 2009)and in a long-term maize monoculture in Italy (Bedini et al., 2007),but lower than those (3.87e3.94 mg/g) in Manawatu and Ngamokograssland soils in New Zealand (Rillig et al., 2006). A range of0.5e0.8 mg T-BRSP/g soil (Table 1) is comparable with that in Texasgrass lands (<2.0 mg, Wright and Upadhyaya, 1998), but is muchlower than that found in grassland soils (9.3e12.0 mg, Rillig et al.,2006; Bedini et al., 2007), Mexican montane rainforest soils(1.0e12.2 mg, Violi et al., 2008) and Costa Rica tropical rainforestsoils (3.9 mg, Lovelock et al., 2004). This may be due to a higherproportion of Glomus species in specific habitats (Treseder andTurner, 2007) or different land uses (Wang et al., 2011). Thus,BRSPs vary with soil types and/or host species. On the other hand,the concentration of glomalin may be dependent on hyphae turn-over, since glomalin is released to soil when hyphae eventually die

Table 1Spatial changes of soil water-stable aggregates, soil carbohydrates, BRSPs and soil relatPoncirus trifoliata.

Soildepth(cm)

Soil water-stable aggregates (%) Ca

0.25e0.50 mm 0.50e1.00 mm 1.00e2.00 mm >2.00 mm Total Hoexca

0e10 7.4� 3.5a, c 32.7� 2.1a, a 35.1� 1.2g, a 15.5� 3.3a, b 90.6� 3.4a 3.710e20 7.7� 0.7a, d 30.7� 2.4b, b 34.5� 1.3g, a 14.2� 4.3a, c 87.2� 4.5a 2.920e30 6.2� 3.0ab, d 29.9� 2.7b, b 42.6� 1.3a, a 14.0� 3.1a, c 92.7� 5.1a 2.930e40 4.7� 2.6b, d 27.8� 1.8g, b 37.4� 1.8b, a 15.5� 1.0a, c 84.6� 1.9a 2.7

Note: data weremeans� SE (n¼ 4) followed by the same letter among soil depths for thedepth within a row (a, b, c, d) are not significantly different at P< 0.05.

or senesce (Driver et al., 2005). In addition, BRSPs were significantlypositively correlated with AM colonization (P< 0.01, Table 2).

Percentages of >2.00 mm WSA and total WSA were similaramong soil depths, whereas total WSA was significantly higher in1.00e2.00 mm> 0.50e1.00 mm> 2.00 mm> 0.25e0.50 mm sizefor the same soil depth (Table 1). Glomalin stabilizes soil aggregatesthrough a ‘gluing’ action (Wright and Upadhyaya, 1998; Bediniet al., 2009). It is therefore reasonable that BRSPs are significantlypositively correlated with the finest 0.25e0.50 mm WSA(P< 0.05e0.01) (Table 2). In addition, EE-BRSP was significantlynegatively correlated with the 0.50e1.00 mm WSA (P< 0.05)(Table 2). In contrast, T-BRSP was positively correlated with2.00e4.00 and 1.00e2.00 mm WSA under the pot-grown AMP. trifoliata seedlings (Wu et al., 2008). This inconsistency with nocorrelation between BRSPs and 1.00e2.00 or >2.00 mm WSA inthis field study may be due to a better development of AM andglomalin under optimal glasshouse conditions.

Root soluble sugar, soil hot water-extractable and hydrolyzablecarbohydrates were highest at 0e10 cm and higher at 10e20/20e30 cm than at 30e40 cm soil depth (Fig. 1; Table 1). Both EE-BRSP and T-BRSP were significantly positively correlated withroot soluble sugar, soil hot water-extractable or hydrolyzablecarbohydrates (P< 0.05 or 0.01) (Table 2), implying that a certainamount of root and soil C is from these GRSPs. Indeed, w40% ofglomalin-stored C was from mycorrhizal roots (Phillips and Fahey,2005) and up to 5% soil C was accumulated with glomalin deposi-tion, when AM hyphae senesced (Treseder and Turner, 2007).

Activity of soil b-glucosidase or protease was significantlydecreased or increased with the increase of soil depth (Table 1).Both EE-BRSP and T-BRSP were significantly positively correlatedwith soil b-glucosidase (P< 0.01, Table 2), suggesting that GRSP,likes b-glucosidase, may contribute to the release of glucosemaintaining metabolically active microbial biomass in the soil(Martinez-Salgado et al., 2010).

Meanwhile, EE-BRSP and T-BRSP were significantly negativelycorrelated with soil protease (P< 0.01, Table 2), inconsistently withprevious results on T-BRSP, obtained in four land use types (farm-land, grassland, orchard and abandoned land) (Tang et al., 2009).Since GRSP can be rapidly degraded by soil protease (Bai et al.,2009), such inconsistency suggests that GRSP degradation varieswith ecological habitats. Such variations between BRSPs and soilprotease might also be attributed to glomalin itself, because glo-malin is mixture of compounds and not a specific single molecule(Rillig and Steinberg, 2002), and the soil distribution of thesevarious compounds possibly influences the activity of soil protease,especially at 30e40 cm depth.

These results confirm that glomalin released by living mycor-rhizal hyphae not only contributes to rhizospheric C but also to Nsince glomalin is a N-linked glycoprotein (Rillig and Steinberg,

ed enzymes in the rhizosphere of Citrus unshiu cv. Guoqing 1 fruit tree grafted on

rbohydrates (mg/g DW soil) BRSPs (mg/g DW soil) Soil enzymes

t water-tractablerbohydrate

Hydrolyzablecarbohydrate

EE-BRSP T-BRSP Protease(mg Glycine/g DW soil)

b-glucosidase(mg sugarreduced/gDW soil)

� 0.2a 16.4� 0.8a 0.6� 0.1a 0.8� 0.1a 0.2� 0.1g 1.5� 0.0a� 0.3b 11.1� 1.9b 0.6� 0.1ab 0.8� 0.1a 0.4� 0.1b 1.0� 0.1b� 0.2b 10.9� 0.5b 0.5� 0.0b 0.6� 0.1b 0.4� 0.1b 0.8� 0.1g� 0.3b 7.8� 1.1g 0.3� 0.0g 0.5� 0.0g 0.9� 0.1a 0.6� 0.1d

same particle size within a column (a, b, g, d) or among particle sizes for the same soil

Table 2Pearson correlation coefficients between EE-BRSP or T-BRSP and other variables (n¼ 16 for the four soil depths).

Mycorrhizalcolonization

Soil water-stable aggregate (mm) Rootsolublesugar

Soil hot water-extractablecarbohydrate

Soilhydrolyzablecarbohydrate

Soil protease Soil b-glucosidase

0.25e0.50 0.50e1.00 1.00e2.00 >2.00 Total

EE-BRSP 0.876** 0.677** �0.614* �0.296 0.182 �0.261 0.882** 0.494* 0.759** �0.889** 0.761**T-BRSP 0.850** 0.502* �0.405 �0.439 0.125 �0.327 0.874** 0.489* 0.778** �0.834** 0.836**

Note: * P< 0.05. ** P< 0.01.

Q.-S. Wu et al. / Soil Biology & Biochemistry 45 (2012) 181e183 183

2002; Lovelock et al., 2004). Therefore, both soil b-glucosidase andprotease should be considered for studying soil C pools.

In conclusion, our results demonstrate that the spatial distri-bution of GRSP is positively affected by mycorrhization, root andsoil carbohydrates and soil b-glucosidase, but negatively by soilprotease.

Acknowledgements

This study was supported by the Key Project of Chinese Ministryof Education (211107), the Science-Technology Research Project ofHubei Provincial Department of Education, China (Q20111301), andthe National Natural Science Foundation of China (30800747). Weare grateful to the Chief Editor RG Joergensen and two anonymousreviewers for their language editing and valuable comments on thismanuscript.

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