Soil Biology & Biochemistry 45 (2012) 181e183

Contents lists available at SciVerse ScienceDirect

Soil Biology & Biochemistry
journal homepage:

Short communication

Spatial distribution of glomalin-related soil protein and its relationships with root mycorrhization, soil aggregates, carbohydrates, activity of 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

College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei 434025, China State Centre of Excellence for Ecohydrology, Edith Cowan University, Joondalup, WA 6027, Australia Centre for Ecosystem Management/School of Natural Sciences, Edith Cowan University, Joondalup, WA 6027, Australia d School of Plant Biology, University of Western Australia, Crawley, WA 6009, Australia
b c

a r t i c l e i n f o
Article history: Received 14 June 2011 Received in revised form 18 August 2011 Accepted 9 October 2011 Available online 20 October 2011 Keywords: b-glucosidase Citrus Glomalin Glomalin-related soil protein Mycorrhization Protease

a b s t r a c t
Relationships between the spatial distributions of glomalin-related soil protein (GRSP) and soil aggregates, carbohydrates or relevant enzymes are poorly studied. We found that two categories of GRSP, the easily extractable Bradford-reactive soil protein (EE-BRSP) and total BRSP (T-BRSP), respectively ranged between 0.3e0.6 and 0.5e0.8 mg/g DW soil, and these two BRSPs decreased with the increase of soil depth (0e40 cm) in the rhizosphere of a 22-year-old Citrus unshiu orchard. Both EE-BRSP and T-BRSP were significantly positively correlated with mycorrhization, 0.25e0.50 mm soil water-stable aggregates, water-extractable or hydrolyzable carbohydrates, and b-glucosidase, but significantly negatively correlated with protease. Our results demonstrate that the spatial distribution of GRSP is significantly affected by mycorrhization, soil carbohydrate, b-glucosidase and protease. Ó 2011 Elsevier Ltd. All rights reserved.

As an insoluble N-linked glycoprotein with w37% carbon (C) and 3e5% nitrogen (N), glomalin contributes to soil structure stabilization and C pools (Wright and Upadhyaya, 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 in short-term experiments (Driver et al., 2005). Less information is available to the spatial distributions of GRSP in the soil profile and its relationships with root and soil carbohydrates, though glomalin is used to evaluate soil quality (He et al., 2010). Soil b-glucosidase is a useful soil quality indicator related to C cycling (Stott et al., 2010), whereas soil protease is highly correlated with soil N transformation (Rejsek et al., 2008). However, it is unclear if soil b-glucosidase or protease is related to GRSP, which contains 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

* Corresponding author. Tel./fax: þ86 716 8066262. E-mail address: (Q.-S. Wu). 0038-0717/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2011.10.002

GRSP and relevant soil variables, we first assessed the spatial distribution of GRSP, carbohydrates, water-stable aggregates (WSA), b-glucosidase and protease in the rhizosphere of Citrus unshiu (cv. Guoqing-1 grafted on Poncirus trifoliata), and then correlated GRSP with these variables. The experiment was conducted in a campus orchard that has w400 22-year-old citrus trees belonging to Yangtze University, China. Sixteen similar plants were randomly selected from four blocks (4-trees  4-blocks). Soils (Xanthi-udic ferralsol) and roots were collected at 0e10, 10e20, 20e30 and 30e40 cm depth within a 2 m radius of tree canopy on 26 May 2010. Samples from 4-trees/ block were mixed as one composite sample. Part of fresh samples was for mycorrhization and enzyme analysis. The rest was air-dried and ground (4 mm) for other analysis. The GRSP is assayed by the Bradford method as Bradfordreactive soil proteins (BRSPs), which include the easily extractable BRSP (EE-BRSP) and total BRSP (T-BRSP) (Rosier et al., 2006). Determination of EE-BRSP and T-BRSP was carried out following Bedini et al. (2009), WSA following Yan (1988), soluble sugar following Yemm and Willis (1954), soil hot water-extractable and hydrolyzable carbohydrates following Li et al. (2002), b-glucosidase activity following Stott et al. (2010), soil protease following

50e1.5 Æ 0. In contrast.9 mg.3g. 2007). Data (means Æ SE.6 mg EE-BRSP/g soil in the rhizosphere of C.7b...00e4.01.6 Æ 3.2g.1a. a 30. (1982).50 mm WSA (P < 0. Violi et al.5 Æ 3. d 4. Root AM colonization was significantly negatively correlated with 0. 2009).6 Æ 0.00 mm > 2..8 Æ 1.5b 7. Wright and Upadhyaya. 2006).00 mm WSA under the pot-grown AM P. grassland.0 mg. . b 29. 2007). open bars) and root soluble sugar concentration (mg/g DW.67e0. inconsistently with previous results on T-BRSP.. d 6.7 Æ 2.8g. g.05e0.0ab. c.00 mm 1. 14.4 Æ 0.2 Æ 0.4 Æ 3. Indeed.7 Æ 5.3b 2.5a. 2009).1b 0.3a.05). P < 0. and EE-BRSP was significantly positively correlated with T-BRSP (r ¼ 0. b c c c 90. P < 0. These results confirm that glomalin released by living mycorrhizal hyphae not only contributes to rhizospheric C but also to N since glomalin is a N-linked glycoprotein (Rillig and Steinberg. solid bars) in the rhizosphere of 22-year-old Citrus unshiu. w40% of glomalin-stored C was from mycorrhizal roots (Phillips and Fahey.7 Æ 2.. a 42. T-BRSP.5 Æ 1.2 Æ 4.8 Æ 0.93.4b. and the soil distribution of these various compounds possibly influences the activity of soil protease. BRSPs vary with soil types and/or host species.94. Soil depth (cm) Soil water-stable aggregates (%) 0. EE-BRSP.5a 92..2 Æ 3. which is consistent with results from C.0 Æ 3. Mexican montane rainforest soils (1.1a 0. 2008) and Costa Rica tropical rainforest soils (3. Percentages of >2. b 35. 2004).0 mm size soil WSA and protease (r ¼ À0. In addition. Both EEBRSP and T-BRSP were significantly positively correlated with root soluble sugar. Spatial distribution of root mycorrhizal colonization (%.00e2.0 mg.1 Æ 1.9 Æ 2. trifoliata seedlings (Wu et al.1g 0. but significantly positively correlated with root soluble sugar.6 Æ 1. such inconsistency suggests that GRSP degradation varies with ecological habitats.2a 2.05.2b 2. Such variations between BRSPs and soil protease might also be attributed to glomalin itself.7 Æ 2.5 Æ 0. but is much lower than that found in grassland soils (9.4 Æ 1. soil hot water-extractable and hydrolyzable carbohydrates. b 27.8 Æ 0. a 37.8a 11.0 Æ 0. 2007) or different land uses (Wang et al. aurantium and P.7 Æ 0. Thus..50 mm 0.00e2. the concentration of glomalin may be dependent on hyphae turnover. 1.1ab 0. Rillig et al. Both EE-BRSP and T-BRSP were significantly positively correlated with soil b-glucosidase (P < 0.0g 0. It is therefore reasonable that BRSPs are significantly positively correlated with the finest 0. soil carbohydrates.94 mg/g) in Manawatu and Ngamoko grassland soils in New Zealand (Rillig et al. BRSPs were significantly positively correlated with AM colonization (P < 0.9a 16.6 mg/g) found in the Mu Us sandland.0b 0. a 15.1b 0.1a 1. 1983). d) are not significantly different at P < 0. unshiu ranked at 0e10 > 10e20 z 20e30 > 30e40 cm soil depth (Fig. This may be due to a higher proportion of Glomus species in specific habitats (Treseder and Turner. orchard and abandoned land) (Tang et al.1b 0. Table 1).6 Æ 0. c 7.00 mm WSA (P < 0. This inconsistency with no correlation between BRSPs and 1.00e2. 1. T-BRSP was positively correlated with 2.00 or >2.4a 87.01). 1998).2 mg. On the other hand.1g 0. A range of 0.7a.01).3e12. EE-BRSP was significantly negatively correlated with the 0. Significantly higher root AM mycorrhization of C. whereas total WSA was significantly higher in 1.0e12.5 Æ 0.57e0. 2002).00 mm WSA in this field study may be due to a better development of AM and glomalin under optimal glasshouse conditions.0a 1.00 and 1.01.01) (Table 2).3 Æ 0.. soil hot water-extractable or hydrolyzable carbohydrates (P < 0.05) (Table 2).9 Æ 0.50e1.00 mm WSA and total WSA were similar among soil depths. BRSPs and soil related enzymes in the rhizosphere of Citrus unshiu cv.0a.3b Hydrolyzable carbohydrate EE-BRSP T-BRSP Root soluble sugar concentration 60 or senesce (Driver et al. trifoliata  C.1a 0..50e1. 15.3e0. Root soluble sugar. 1). c) above the bars are significantly different among soil depths for each variable at P < 0. but lower than those (3. since glomalin is released to soil when hyphae eventually die Table 1 Spatial changes of soil water-stable aggregates.89. may contribute to the release of glucose maintaining metabolically active microbial biomass in the soil (Martinez-Salgado et al.4 Æ 0.3a. / Soil Biology & Biochemistry 45 (2012) 181e183 80 80 a b a b b c b c 70 70 60 50 40 30 20 10 0 50 40 30 20 10 0 0-10 10-20 20-30 30-40 Soil depth (cm) Fig.. soil hot water-extractable and hydrolyzable carbohydrates were highest at 0e10 cm and higher at 10e20/ 20e30 cm than at 30e40 cm soil depth (Fig.05.8 mg T-BRSP/g soil (Table 1) is comparable with that in Texas grass lands (<2. 2008). when AM hyphae senesced (Treseder and Turner. 2006. likes b-glucosidase.0g Note: data were means Æ SE (n ¼ 4) followed by the same letter among soil depths for the same particle size within a column (a. Activity of soil b-glucosidase or protease was significantly decreased or increased with the increase of soil depth (Table 1).. Bedini et al. because glomalin is mixture of compounds and not a specific single molecule (Rillig and Steinberg. and AM colonization following Phillips and Hayman (1970).2 Æ 4. 2005)..8 Æ 0.1d 0e10 10e20 20e30 30e40 7. Both EE-BRSP and T-BRSP were highest at 0e10 and 10e20 cm and higher at 20e30 cm than at 30e40 cm soil depth (Table 1). EE-BRSP and T-BRSP were significantly negatively correlated with soil protease (P < 0. 2010). Since GRSP can be rapidly degraded by soil protease (Bai et al. Table 2).3e0. 2011). Table 2).6 Æ 1.05 or 0.9 Æ 0.6 Æ 0. unshiu (Table 1) is consistent with those (0. 14. Meanwhile.5e1. 2009). b. suggesting that GRSP.00 mm Total Carbohydrates (mg/g DW soil) BRSPs (mg/g DW soil) Soil enzymes Hot waterextractable carbohydrate 3. Root mycorrhizal colonization b-glucosidase Protease (mg Glycine/ (mg sugar g DW soil) reduced/g DW soil) 0.87e3.1 Æ 1. d 32..9 Æ 0.25e0.3a.25e0. b. Table 2).8 Æ 1. Wu et al.01. P < 0.7 Æ 0. especially at 30e40 cm depth.00 mm >2. Lovelock et al. Glomalin stabilizes soil aggregates through a ‘gluing’ action (Wright and Upadhyaya.. b.1a 84.5 Æ 1.50 mm size for the same soil depth (Table 1). a 34.1b 0. 2007). obtained in four land use types (farmland.01) (Table 2). d) or among particle sizes for the same soil depth within a row (a.6 Æ 0. and soil b-glucosidase (r ¼ 0.00 mm > 0. China (Bai et al.9b 10..00 mm > 0. n ¼ 4) followed by different letters (a. Guoqing 1 fruit tree grafted on Poncirus trifoliata.5e0.1g 0.6b. In addition.7 Æ 0.4 Æ 0.-S.1a.182 Q. implying that a certain amount of root and soil C is from these GRSPs. 2009) and in a long-term maize monoculture in Italy (Bedini et al. 2005) and up to 5% soil C was accumulated with glomalin deposition. Bedini et al.00e2.25e0.8b.1a 0. limon (Levy et al. A range of 0.9 Æ 0. 1998. Cao et al.

Escamilla-Prado. D.. Agricultural Press. Estimation of protease activity in soils at low temperatures by casein amendment and with substitution of buffer by demineralized water. 421e428 (in Chinese with English abstract). Yemm. Andrews. Zhao.. Arbuscular mycorrhizal development. 276e290. Wang.182 0.. glomalin and soil enzymes under the canopy of Astragalus adsurgens Pall.J.. Rosier. Hoye. R.. S. Rejsek. R. Effect of irrigation. He. Acknowledgements This study was supported by the Key Project of Chinese Ministry of Education (211107). H. Menge.S.. 2010.-S. Xia.P. Zhao. C. Forest Ecology and Management 254. M.. Giovannetti.614* À0.J..D. P. in Mu Us sandland. 2967e2970.L. 2006.. Soil Biology and Biochemistry 37. 2008. . Singapore. 417e425. Y. Transactions British Mycological Society 55.261 À0. X.. R.. B. Upadhyaya.W. A. M.. 1371e1374. Minimal direct contribution of arbuscular mycorrhizal fungi to DOC leaching in grassland through losses of glomalinrelated soil protein. Spatial distribution of arbuscular mycorrhizal fungi.N. 2007.. A survey of soils for aggregate stability and glomalin.. China. Willis.C. Patterns of rhizosphere carbon flux in sugar maple (Acer saccharum) and yellow birch (Betula allegheniensis) sapling. A. Argese. Xia. but negatively by soil protease.. K. 411e417. glomalin-related soil protein (GRSP) content. Ba. B. Dynamics of arbuscular mycorrhizal fungi and glomalin in the rhizosphere of Artemisia ordosica Krasch..L... H.. K. Mycorrhizal colonization EE-BRSP T-BRSP 0.50 0. 1983... Research Methods of Soil Fertility (in Chinese).P.T.X. 107e119.. 122e128.. Improved soil structure and citrus growth after inoculation with three arbuscular mycorrhizal fungi under drought stress. S. 1998. 1257e1266.M. and rhizospheric phosphatase activity in citrus orchards under different types of soil management.W. Soil Biology and Biochemistry 42.B.D. L. Martinez-Salgado. E. Karlen. Pte. M.Y. 2002. Soil Science Society of America Journal 71. Q.125 Total À0..G. Mexico. Avio... Global Change Biology 11. pp. C.. Biological soil quality indicators: a review. In: Mendez-Vilas.. our results demonstrate that the spatial distribution of GRSP is positively affected by mycorrhization. S. Wang. Liu.. root and soil carbohydrates and soil b-glucosidase. Clark. China. Y.Q. Ortega-Blu. Dodd.E. T. C. A.J. and the National Natural Science Foundation of China (30800747)..761** 0.. Z. European Journal of Soil Biology 44. ** P < 0. E. W..502* 0.. Wang. J.882** 0.. Carran. 39e40 (in Chinese with English abstract).C. 278e287.. Wright. 2010. Avio. Lovatt. Bedini. Ruess. Phillips... Disturbance changes arbuscular mycorrhizal fungi phenology and soil glomalin concentrations but not fungal spore composition in montane rainforests in Veracruz and Chiapas.01.. Improved produres for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection..S. both soil b-glucosidase and protease should be considered for studying soil C pools. L.. Journal of Ecology 92. J. Biochemical Journal 57.. Changes in soil aggregation and glomalin-related soil protein content as affect by the arbuscular mycorrhizal fungi species Glomus mosseae and Glomus intraradices...Q. Glomalin production by an arbuscular mycorrhizal fungus: a mechanism of habitat modification. V.. C..S... X. 2006. References Bai. He. Phillips. J.X. Li.. T. J. 2011. M. Ltd. Steinberg.. S. Journal of Plant Nutrition and Soil Science 174.E. X. osmotic adjustment and photosynthesis of citrus under well-watered and water stress conditions. Cui.J. / Soil Biology & Biochemistry 45 (2012) 181e183 Table 2 Pearson correlation coefficients between EE-BRSP or T-BRSP and other variables (n ¼ 16 for the four soil depths).C.M.C. Bazzoffi. 983e995. M.850** Soil water-stable aggregate (mm) 0. M. 508e514. Liu. Ecosystems and Environment 120. R. Liebig.A.. Glomalin in ecosystems.. Violi. water salinity and rootstock on the vesicular distribution of vesicular-arbuscular mycorrhiza in citrus roots.S.00e2. 2004.494* 0. Arbuscular mycorrhizal fungi influence growth.. L. Shan. Rillig. Agriculture.. S.. Krikun.. The estimation of carbohydrates in plant extracts by anthrone. Chinese Journal of Soil Science 13.L. R. 2005. J. L. Soil Biology and Biochemistry 41. Pellegrini. C. 101e106.L. Lovelock. R.). 397e403. A. Q. A.25e0. Therefore..F.J.. Fahey. 2007.00 0. 2008. 2002.L.00 À0... Driver. M.. Effect of land use type on profile distribution of glomalin.. Morton. Formanek. 2002.. A. Wright.L.. Z.X. Amino Acids 35.T.. Comprasions of determined methods of several soil protease activities. S. Zhou. Wang. 941e947. Evaluation of b-glucosidase activity as a soil quality indicator for the soil management assessment framework. Rillig.A. Effects of long-term land use on arbuscular mycorrhizal fungi and glomalin-related soil protein. 1491e1496. Tang. Treseder. L... Lovelock et al. P.836** Note: * P < 0. Bedini.876** 0. Chinese Journal of Eco-Agriculture 17. J. a glycoproteins produced by hyphae of arbuscular mycorrhizal fungi.05.874** Soil hot waterextractable carbohydrate 0. L.. 2205e2211. Soil Biology and Biochemistry 38... 2009. (Ed. Turner.50e1. We are grateful to the Chief Editor RG Joergensen and two anonymous reviewers for their language editing and valuable comments on this manuscript.. Jannsens.F. A. Dong. H. 463e466.Y.E.S.M. Technology and Education Topics in Applied Microbiology and Microbial Biotechnology...K.. D. Giovannetti. D. Hayman. Gutiérrez-Romero.F. 1313e1319. Hoye. Yan. Glomalin-related soil protein: assessment of current detection and quantification tools. Levy. Soil stocks of glomalin produced by arbuscular mycorrhizal fungi across a tropical rainforest landscape. Stott. 158e161. 2009. Li. L. 1982. Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. 319e328. Y. 2008.439 >2.00 À0. K. P. the Science-Technology Research Project of Hubei Provincial Department of Education. M. Xia.M. 2009. L. 97e107.F. Holben. 1970. Tang. 1954. 2006. C. 2005... D. 2004).. Cao.677** 0.L.L.. New Phytologist 95.H. Beijing. J. In conclusion.K.A. Pellegrino.. Q.M. Soil Biology and Biochemistry 38... Argese. Soil Science Society of America Journal 74.296 À0. 1988.J. S. 65e72. Zhang.405 1.834** 0. E.A.778** Soil protease 183 Soil b-glucosidase À0..327 Root soluble sugar 0. Wu.759** 0. Plant and Soil 198. Wu et al. M.. World Scientific Publishing Co..889** À0. Soil Biology and Biochemistry 34.489* Soil hydrolyzable carbohydrate 0. Barrientos-Priego. Rillig. China (Q20111301). Wu.. Rillig. C...R. Wienhold.. Soil Biology and Biochemistry 41. Zou. E. in the Mu Us sandland. Pavelka.J. Journal of Plant Physiology 163. Wu. Wright. 1137e1142 (in Chinese with English abstract). P. Acta Pedologica Sinina 39.... M. Effect of straw on soil organic carbon constitution and structural stability. E. 2010. Current Research. M.