Rhizosphere microbial diversity as influenced by humic substance amendments and chemical composition of rhizodeposits

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Abstract

The influence of humic substances (HS) on crops and soil microbial communities still lacks a complete understanding. The influence of HS on chemical composition of maize rhizodeposits and diversity of rhizosphere microbial communities was studied here in rhizobox systems. Eleven different HS were characterized by elemental analyses and 13C-CPMAS NMR, and applied to the upper compartment of rhizobox systems cultivated with maize. Once a full rhizoplane was formed, the lower compartment of the rhizoboxes was sampled with a slicing device, in order to obtain rhizopshere (0–2 mm) and bulk (> 10 mm) soil samples, which were subjected to chemical analyses of organic acids and sugars (pentoses and hexoses), bioassays with a luminescent biosensor for the detection and quantification of bioavailable C forms, and analyzed for bacterial diversity by means of PCR-DGGE fingerprint. Multivariate techniques were applied to assess correlations between the parameters involved and to assess the most relevant chemical drivers of rhizosphere microbial diversity. Results showed that the hydrophilic and poorly aromatic HS changed the pattern of sugars and organic acids released in the rhizosphere by maize roots, and this, in turn, affected the composition of rhizosphere microbial communities. Differences in chemical and microbiological composition were also found, but to a lesser extent, among bulk soil samples of different treatments, thus indicating that rhizodeposits can influence soil composition at several mm distance from the root plane. Multiple regression analyses suggested a prominent role of succinic and malic acid in shaping the diversity of microbial communities. The approach and results described here proved to be useful to test bioactivity of HS towards an agricultural crop, and identify correlations between chemical and microbiological constituents at the tri-phase soil–roots–microbes system.

Introduction

The rhizosphere is a hot spot of soil fertility, where complex interactions between plant roots, microorganisms and soil determine the turnover of organic materials and the availability of nutrients for plant growth (Badri et al., 2009). The complexity of the tri-phase soil–plant–microbes system is further increased by the wide range of soil organic and inorganic constituents, some of which can exert bio-active effects towards plants and microorganisms. In particular, humic substances (HS) are among the most complex and biological active compounds in soil (Nebbioso and Piccolo, 2011), and are known to stimulate in a number of ways both plant and microbial activities (Kulikova et al., 2010, Piccolo et al., 1992, Pinton et al., 1992, Nardi et al., 1996, Nardi et al., 2002b;). In the case of plants, direct and indirect effects of HS on their physiology have been demonstrated. Indirect effects include induced changes in soil physic-chemical properties, in the structure and activity of soil microbial communities or in the availability of nutrients, trace elements or growth regulators (Nardi et al., 1996, Varanini and Pinton, 2001). Direct effects are instead a consequence of the flow of HS into the apoplast, and they may include induction of H+-ATPase synthesis and transport proteins, hormone-like effects and effects on glycolysis and enzymes involved in the Krebs cycle (Nardi et al., 2002a, Pinton et al., 1992, Pinton et al., 1997). Negative effects of HSs in the rhizosphere have also been reported, and have been explained by non-chemical mechanisms, i.e., reduction of roots hydraulic conductivity through physical interactions between HS and cell wall pores (Asli and Neumann, 2010).

Microorganisms are also interacting with HS in soil and rhizosphere environments in a number of ways. HS have stimulating effects on amylolitic, proteolytic and denitrifying microorganisms (Visser, 1985), and have the ability of crossing the membrane of microorganisms and bioconcentrate within the cells (Kulikova et al., 2010). HS are also known to influence deeply the structure and activity of soil microbial communities (Dong et al., 2009, Puglisi et al., 2009) and to select phylogenetic distinct and abundant guilds, as in the case of microbial groups involved in nitrate reduction processes that are directly linked to the oxidation of the humic material (Van Trump et al., 2011).

A general consensus considers HS as supramolecular associations of relatively small (< 1000 Da) hetereogeneous molecules, which are held together in only apparently large molecular sizes by weak linkages, such as hydrogen and hydrophobic bonds (Piccolo, 2002). The main molecules that constitute the “bricks” of these supramolecular associations are numerous, and belong to several chemical classes such as organic acids, phenols, sugars, and proteins (Nebbioso and Piccolo, 2011, Nebbioso and Piccolo, 2012). Aliphatic, carboxylic and aromatic C forms dominate in different proportions, thus differentiating the chemical properties of HS of different origins (Canellas et al., 2010). This supramolecular structure of HS is not only confirmed by several experimental evidence (Liu et al., 2011, Peuravuori, 2005, Piccolo, 2002, Piccolo et al., 2002, Smejkalova and Piccolo, 2008), but is also coherent with their bioactivity, which would be mostly unlikely assuming a macropolymeric structure stabilized and crosslinked by covalent bonds.

Since HS influence both plants and microbes in a number of ways, their addition to soil (e.g. through compost or other organic fertilizer) can affect the complex interactions already occurring between microorganisms and plant roots (Bais et al., 2004), being rhizodeposition one of the most important of these interactions. Through rhizodeposition, plants release significant amounts of C in the rhizosphere (10 to 45 % of net assimilated C). In turn, microbes support plants by mobilizing and making more available the nutrients required by plants, especially nitrogen, although it has been demonstrated that also low-molecular weight compounds can be deposited by microbes and then uptaken by plants (Jones et al., 2009). This mutual interaction between microorganisms and roots has been described as the “microbial loop” (Bonkowski, 2004, Paterson, 2003) and is a key factor in determining soil fertility.

Previous works have demonstrated that addition of either compost, hydrophilic and hydrophobic fractions of dissolved organic matter (DOM), or humic acids of different size fractions, can have a significant effect on the amount of bioavailable C deposited by maize plant roots, thus resulting in a significant change in the structure of soil microbial communities (Puglisi et al., 2008, Puglisi et al., 2009). The aim of this work was to better elucidate the interactions occurring among HS chemical properties, total amount of bioavailable C deposited by maize roots when amended with such HS, chemical composition of rhizodeposits, and structure of rhizosphere microbial communities. The main objectives of the work were: i) to confirm that HS can alter the quantity and quality of low-molecular weight C compounds deposited by roots and, in turn, the structure of rhizosphere microbial communities; ii) to recognize the possible correlations between the molecular structure of humic and fulvic acids and rhizodeposits chemical composition; and iii) to identify which components of rhizodeposits are most responsible for the observed changes at microbial community level.

Section snippets

Soil

An orchard loamy soil was collected near Matera, South-East Italy. Soil samples (7 cm diameter cores) were randomly collected from the surface layer (0–20 cm) at least 2 m far from trees, sieved at 2 mm, pooled on site and stored at 4 °C before using for bulk soil microcosms and rhizobox experiments. Soil had a loam texture (24.7% clay, 33.8% silt 41.5% sand), a pH (H2O) of 6.7 and total organic C of 8.3 mg C g 1 d.w. soil, with approximately 50% of total organic carbon being represented by humic

Molecular characteristics of humic substances

A different molecular composition for the selected HS was revealed, by the results of both elemental analyses and 13C-CPMAS NMR spectra (Table 1). A large predominance of aromatic and alkyl-aromatic components was found in the NMR spectra of HA-A, HA-B and HA-C samples. The aromatic-C (110–160 ppm) signals accounted, for 52, 47 and 85% of the total area in the NMR spectra of these respective humic acids. Moreover the resonances of methylene chains and methyl groups in the alkyl-C region (0–45 

Discussion

Soil is indeed an environment extremely rich in chemical compounds and biological species, mainly microorganisms (Dance, 2008). This complexity is further enhanced in the rhizosphere due to the large number of biochemical compounds released by plant roots, which in turn stimulate a differentiation of microbial activity, and an intense chemical dialogue between plant roots and microbes, whose decoding is only at its infant stages (Badri et al., 2009). A deeper understanding of rhizosphere

Conclusions

The present work was conducted with the aim of better elucidating the effects of HS on the rhizodeposition of maize plants. A consolidated experimental set-up was applied in order to assess effects of HS mediated by changes in plant physiology. It was found that HS can indeed change the nature and amount of low molecular weight compounds released by amended plants, but these effects depend from the nature of the HS applied. It was then found that in all cases the structure of microbial

Acknowledgments

This research has been supported by the project Structure-activity relationships of natural organic matter in soil-plant systems" funded by MIUR (Italian Ministry for University and Research) and by the project "Synthetic and natural agro-chemical compounds: ecological impacts on the soil ecosystem and effects on plant production", sponsored by Cariplo Foundation, Italy.

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