Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices

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Abstract

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration – compared to initial values – in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1–2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized.

The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.

Introduction

Arbuscular mycorrhizal (AM) fungi are mutualistic symbionts living in association with the roots of the majority of land plants. They are key organisms of the soil/plant system, influencing soil fertility and plant nutrition (Smith and Read, 2008). The large network of fungal hyphae, which spreads from mycorrhizal roots into the surrounding soil, affects the physico-chemical characteristics of soils and represents stabilizing agents in the formation and maintenance of soil structure (Miller and Jastrow, 2000). Many reports have shown that AM fungi are able to counteract soil erosion by increasing the stability of soil aggregates (Andrade et al., 1998, Bethlenfalvay et al., 1999, Miller and Jastrow, 2000) through the combined action of extraradical hyphae and their exudates and residues (Gupta and Germida, 1988, Tisdall and Oades, 1982, Miller and Jastrow, 1990, Miller and Jastrow, 1992). Among these fungal components, glomalin, an insoluble and hydrophobic proteinaceous substance (Wright et al., 1996), is of particular interest. Glomalin has been proposed to improve the stability of soil by avoiding disaggregation by water (Wright and Upadhyaya, 1998, Zhu and Miller, 2003, Wright et al., 2007). A strong relationship between glomalin concentration and the amount of water stable aggregates (WSA) has been demonstrated (Wright and Upadhyaya, 1998, Rillig et al., 2001, Harner et al., 2004, Rillig, 2004). Moreover, previous research showed that the proportion of WSA higher than 1–2 mm size class is a highly sensitive indicator of the effects of different cropping systems and management practices on soil structure stability (Topp et al., 1997, Marquez et al., 2004).

Glomalin has been detected and quantified in different soil free cultivation systems (Rillig and Steinberg, 2002, Driver et al., 2005, Gadkar and Rillig, 2006), by using a specific monoclonal antibody in ELISA or by Bradford assay. The latter method is also utilized to evaluate glomalin in soil, where it accumulates because of its low turnover rate (Steinberg and Rillig, 2003). However, it has been proposed that the product of soil extraction (121 °C in citrate buffer) evaluated by the Bradford assay represents a proteinaceous material, named glomalin-related soil protein (GRSP), rather than glomalin (Rillig, 2004), since such assay may detect also proteins originated from sources other than AM fungi (Rosier et al., 2006). In addition, recent reports (Jonathan and Javier, 2006, Schindler et al., 2007, Whiffen et al., 2007) have shown that polyphenolic compounds, such as soil tannins and humic acids, may be co-extracted with glomalin and interfere with the Bradford quantification. Thus, in previous field studies on the effect of mycorrhizal symbiosis on soil glomalin concentration (Wright and Upadhyaya, 1998, Rillig et al., 2001, Lutgen et al., 2003), erratic organic matter inputs may have affected data on AM fungal contribution to soil glomalin concentration. As a consequence, although glomalin is assumed to be produced by AM fungi, the relationship between GRSP and AM fungal occurrence remains to be verified.

With the aim of investigating such relationship, and considering that glomalin is probably released in soil by AM fungal extraradical mycelium (Driver et al., 2005), we carried out microcosm experiments with four AM fungal isolates, which had been previously characterized for their ability to develop extraradical mycelium (ERM) showing differences in the extent, structure and interconnectedness of mycelial networks (Avio et al., 2006).

Here we assessed (i) the relationship between mycorrhizal establishment in Medicago sativa plants, inoculated with different species and isolates of AM fungi, and GRSP concentration, (ii) the effects of different AM fungi on soil quality variables, such as soil aggregate stability and soil organic matter (SOM), (iii) the relationship between ERM variables of AM fungal isolates and aggregate stability and GRSP concentration.

Section snippets

Plant and fungal material

AM fungi used were: Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe, isolate IMA1 from UK (collector B. Mosse) and isolate AZ225C from USA (collector J. C. Stutz), and Glomus intraradices Schenck & Smith, isolate IMA5 from Italy (collector M. Giovannetti) and isolate IMA6 from France (collector V. Gianinazzi-Pearson). They were obtained from pot cultures maintained in the collection of the Soil Microbiology Laboratory of the Department of Crop Plant Biology, University of Pisa, Italy. The

Soil variables

Soil GRSP contents of mycorrhizal pots, measured as EE-GRSP and T-GRSP, showed large increases compared with the original soil mixture (0.29 ± 0.04 and 1.08 ± 0.04 mg g−1 dry soil respectively) (Fig. 1). After four months' growth, mean increases of EE-GRSP and T-GRSP in mycorrhizal pots were 32.4 and 34.9%, respectively, compared to soil mixture values (EE-GRSP, P < 0.001; T-GRSP, P < 0.001). On the contrary, GRSP contents, both EE-GRSP and T-GRSP, of non-mycorrhizal pots did not show

GRSP production by mycorrhizal and non-mycorrhizal plants

Mycorrhizal treatments produced an increase in the initial value of GRSP concentration in pot soil, compared with non-mycorrhizal pots, suggesting a cause-effect relationship between mycorrhizal symbiosis and GRSP content. Our data are direct evidences of differences in GRSP production by mycorrhizal and non-mycorrhizal plants. Many circumstantial evidences, reviewed by Rillig (2004), showed the active role of AM fungi in GRSP production. Positive links between GRSP and AM fungal biomass were

Conclusion

The results of our work show that: (i) mycorrhizal establishment in M. sativa plants inoculated with different species and isolates of AM fungi produced an increase in GRSP concentration – compared to initial values – in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1–2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP

Acknowledgments

This work was supported by MIUR, FISR 2002, project SOILSINK “Cambiamenti climatici e sistemi produttivi agricoli e forestali: impatto sulle riserve di carbonio e sulla diversità microbica del suolo”.

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