Plant Soil (2012) 359:149–163 DOI 10.1007/s11104-012-1198-3
REGULAR ARTICLE
Investigation of organic anions in tree root exudates and rhizosphere microbial communities using in situ and destructive sampling techniques Shengjing Shi & Maureen O’Callaghan & E. Eirian Jones & Alan E. Richardson & Christian Walter & Alison Stewart & Leo Condron
Received: 11 November 2011 / Accepted: 26 February 2012 / Published online: 10 March 2012 # Springer Science+Business Media B.V. 2012
Abstract Purpose Understanding of the role of low molecular weight organic anions (OAs) in structuring rhizosphere microbial communities in situ is limited due to challenges associated with sampling. Improved techniques are needed for such studies. Methods This study used in situ and destructive sampling techniques and compared two exudate extraction methods [anion exchange membrane (AEM) capturing and water extraction] from rhizosphere and
non-rhizosphere samples of genetically modified (GM) and control Pinus radiata D. Don trees grown in large-scale rhizotrons for ~10 months. Metabolically active soil microbial communities were analysed using rRNA-DGGE. Results Recovery of eight out of 12 anions was influenced by extraction methods, and in situ sampling using AEM was shown to be the most efficient method. Only minor differences were detected in OAs in root exudates collected from the GM and control trees. Significant
Responsible Editor: Hans Lambers. Electronic supplementary material The online version of this article (doi:10.1007/s11104-012-1198-3) contains supplementary material, which is available to authorized users. S. Shi : A. Stewart : L. Condron Bio-Protection Research Centre, Lincoln University, PO Box 84, Lincoln 7647, Christchurch, New Zealand
C. Walter Scion—Next Generation Biomaterials, Private Bag 3020, Rotorua, New Zealand
M. O’Callaghan AgResearch, Private Bag 4749, Christchurch 8140, New Zealand A. E. Richardson CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia E. E. Jones : L. Condron Faculty of Agriculture and Life Sciences, Lincoln University, PO Box 84, Lincoln 7647, Christchurch, New Zealand
Present Address: S. Shi (*) Department of Environmental Sciences, Policy and Management, University of California, Berkeley, CA 94720, USA e-mail:
[email protected]
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differences in α-Proteobacterial and Pseudomonas communities were associated with the two tree lines in the topsoil at both sampling events. Additional differences in β-Proteobacterial and fungal communities between tree lines were detected in the rhizosphere using destructive sampling. Conclusion This study demonstrated that in situ sampling was superior to destructive sampling for the efficient collection of root exudates and analysis of associated rhizosphere microbial communities. Keywords Anion exchange membrane . In situ sampling . Organic anions . Pinus radiata . Rhizosphere microbial community . Root exudates Abbreviations OAs Organic anions GM genetically modified AEM anion exchange membrane
Introduction Low molecular weight organic anions (OAs) are a major component of root exudates. As such, they are a significant C source for microorganisms and are an important factor influencing bacterial community structure in the rhizosphere (Bertin et al. 2003; Grayston et al. 1996; Jones 1998; Marschner 1995). Our previous work has demonstrated the role of OAs in shaping bacterial community structure in soils (Shi et al. 2011b). To date only a few studies have attempted to directly link the amounts and nature of root exudates to the structure of rhizosphere microbial communities, as it is acknowledged that there are significant technical challenges associated with sampling exudates and rhizosphere soil (Biedrzycki and Bais 2009; Neumann et al. 2009; Phillips et al. 2008). In addition, most studies have used annual plants with a short life cycle, and have generally relied on destructive sampling techniques. For example, Micallef et al. (2009) collected root exudates of Arabidopsis thaliana grown in hydroponic solutions and analysed rhizosphere bacterial communities associated with roots of A. thaliana grown in soil. However, plants grown in solutions and soil may differ in root morphologies and other rhizosphere processes including exudation (Kamilova et al. 2006; Mucha et al. 2005). Weisskopf
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et al. (2008) studied the link between root exudates in rhizosphere soils and their bacterial communities by removing wheat and lupin root systems from soil, rubbing rhizosphere soil off the roots and extracting OAs from root and soil samples using sterile water. However, such approaches are time consuming and may damage roots. Furthermore, these techniques have limited application for larger plants such as trees which are longerlived and have substantially larger root systems. We previously developed a novel in situ root exudate collection method using anion exchange membrane (AEM) from trees grown in a Biotron system (Shi et al. 2011a). Such an approach has potential for wider application to rhizosphere process studies with linkage to microbial community analysis and offers some advantages over previous destructive sampling-based techniques (Shi et al. 2011a). The primary objective of this study was to compare in situ and destructive sampling of OAs using AEM capture and water extraction methods using radiata pine, Pinus radiata D. Don, as a model system. This species is the dominant commercial plantation forest tree species in New Zealand, mainly because of its rapid growth and high productivity. Genetic modification is being investigated as a means of improving productivity of P. radiata (Lottmann et al. 2010). As with other genetically modified (GM) plants, the prospect of commercial release of GM trees raises concerns regarding potential ecological and environmental impacts (Valenzuela et al. 2006; Walter 2004). In comparison with short-lived agricultural crops, GM trees may remain on a site for many years and thus have potential to significantly impact soil ecosystem processes and microflora (Lamarche and Hamelin 2007; LeBlanc et al. 2007). However, more recent data from field tests with GM trees confirms their environmental safety (Walter et al. 2010; Lottmann et al. 2010; Schnitzler et al. 2010). In studies where differences in the microbial communities associated with GM plants and their unmodified parental lines have been detected, these differences have often been attributed to changes in the composition or quantity of root exudates (Di Giovanni et al. 1999; Milling et al. 2004; Sessitsch et al. 2003). In this study, the composition and quantity of root exudates and the structure of metabolically active rhizosphere microbial communities associated with GM and control pine trees grown in a rhizotron system were examined.
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Materials and methods Rhizotron experiment The experiment was conducted using rhizotrons in the New Zealand Biotron as described by Shi et al. (2011a). Rhizotrons (0.8 m diameter×0.5 m deep) were packed with the A and B horizons of a sieved (10 mm) Templeton sand-loam soil (Immature Pallic soil; New Zealand Soil Bureau 1968) collected from a pasture at Lincoln University, Canterbury, New Zealand. pH values for both horizon A and B soils were 5.6 and CEC values were 11 and 9 me 100 g−1, respectively. Other soil properties and rhizotron packing details are shown in the supplementary information (Table S1). Each rhizotron cylinder was split vertically with a Signex panel to provide two separate 0.25 m2 compartments which were treated as experimental replicates. Clonal Pinus radiata seedlings were developed from cuttings derived from single parental lines that were either unmodified control or GM for expression of leafy and nptII genes (Scion Research, Rotorua, New Zealand) (Lottmann et al. 2010). Single GM and unmodified control seedlings (30–40 cm high; ~1 year old) were planted in each rhizotron compartment, and the experiment comprised four replicates of each tree type. Trees were grown for 10 months under conditions described previously (Shi et al. 2011a). Rhizotrons were rotated 90° every week and randomized within the growth room every 6 weeks during the experimental period. After 3 months growth, horizontal access cores (5 cm diameter x 45 cm long) were removed at depths of 10 and 20 cm to allow for observation and sampling. In situ observations of root growth were achieved using a medical endoscope system (Karl Storz, Germany) as described previously (Shi et al. 2011a). The physical integrity of the access cores was maintained between observations or sampling events using a removable inflatable tube. Exudate and soil sampling In situ sampling Root exudate sampling using AEM was conducted after 9 months growth in rhizotrons when trees were 80–100 cm high. Organic anions present in root and adjacent soil regions were collected from the access portals at the depths of 10 and 20 cm using AEM pre-
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charged with HCO3−and backed with Whatman 3 MM filter paper (Shi et al. 2011a). Three areas of root-soil region were sampled in each access portal using 1× 2 cm strips of AEM-Whatman 3 MM held in place with a thumbtack and re-insertion of the inflatable tube. Exudate solutions were collected from trees in two randomly selected access portals over 2 h between 10 am and midday for 16 successive days. An area of non-rhizosphere soil where no roots or fungal mycelia were observed was also sampled as a “non-rhizosphere” region from each access portal. Following collection, images of the root areas were recorded by digital photography and root areas in the images were calculated using imaging software (Shi et al. 2011a). Collected AEM strips were rinsed with sterile deionised water, transferred to 1.5 ml of 0.5 M HCl and placed at 4°C for 3 h in a shaker at 150 rpm for elution of OAs. The three rhizosphere samples from each access portal were bulked together to form a single sample and stored at −20°C prior to HPLC analysis. Root samples with adhering soil were obtained from the regions used for exudate collection using long-arm scissors and the three samples collected from the same access portals were shaken gently to remove loose (non-rhizosphere) soil before being combined. Non-rhizosphere soil samples (approximately 2 g) were also collected from the regions where nonrhizosphere soil solution had been collected. A total of eight replicates were obtained for each category (rhizosphere soil, non-rhizosphere soil) at each depth (10 and 20 cm) from each tree line (GM and control) resulting in a total of 64 samples. Destructive sampling Destructive sampling was carried out 4 weeks after in situ sampling (10 months) by removal of 10 cm diameter vertical soil cores at a single location ~20 cm from the base of the tree. Soil was sampled at two depths (D100– 14 cm, D2014–28 cm), and roots with associated rhizosphere soil were carefully collected from each soil core and gently shaken to remove loose (non-rhizosphere) soil (Ruark et al. 1991; Tend and Timmer 1995). The remaining soil in the core was considered to be nonrhizosphere soil. Four samples were obtained for each soil category (rhizosphere soil, non-rhizosphere soil) at each depth (D1 and D2) from each tree line (GM and WT), providing a total of 32 samples.
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All the exudate samples were analysed by HPLC targeting 12 common OAs previously shown to be present in soil-root exudates of radiata pine trees (Shi et al. 2011a). Briefly, OAs were separated on a Prevail™ organic acid column (250×4.6 mm, 5 μm particle size; Alltech, USA) using Waters 490 E programmable multiwavelength detector (Waters Pty Ltd, USA). Descriptions of mobile phase components, analytical standards, levels of sensitivity etc are given elsewhere (Shi et al. 2011a).
USA) according to the manufacturer’s instructions (Promega Corporation, WI, USA); random hexamer primers were used for bacterial 16S rRNA and FR1 reverse primer (Vainio and Hantula 2000) for fungal 18S rRNA. A negative control using DNase/RNase-free distilled water (Invitrogen) instead of RNA sample was included in each RT-PCR reaction. Bacterial and fungal communities were assessed following PCR amplification of cDNA templates using general-bacterial 16S primers (Muyzer et al. 1993) and fungal 18S primers (Vainio and Hantula 2000; White et al. 1990). Commonly reported rhizosphere bacterial groups α-Proteobacteria, β-Proteobacteria and Pseudomonas (Chow et al. 2002; Haas and Défago 2005; Lottmann et al. 2010; O’Callaghan et al. 2008) were also investigated in this study to improve the resolution of complex bacterial communities in soils. Amplification conditions and programmes and DGGE conditions are described in detail in the supplementary information (SI, Materials and Methods). DGGE gels were scanned using a GS-800 Imaging Densitometer (Bio-Rad) and bands scored using Quantity One software (version 4.4.1; Bio-Rad) and were saved using Diversity Database (version 2.2.0; Bio-Rad). Peak identification, band clustering and construction of similarity matrix based on the presence/absence of bands were described in detail previously (Clough et al. 2009; Lottmann et al. 2010).
Molecular analysis of soil microbial communities
Statistical analysis
Rhizosphere soil samples were prepared for microbial community analysis as described by Lottmann et al. (2010). Rhizosphere and non-rhizosphere soils were then stored at −80°C prior to analysis. Microbial RNA was extracted from soil samples (up to 0.5 g) by vortexing in Lysing Matrix B tubes (Q-Biogene, Carlsbad, CA, USA) with 0.5 ml of hexadecyl-trimethyl-ammonium bromide buffer followed by a phenol-chloroform purification protocol modified from Griffiths et al. (2000) and described by Clough et al. (2009). Prior to RT-PCR, nucleic acid samples were treated with DNase (TURBO DNA-free™ Kit, Applied Biosystems, CA, USA), and aliquots of samples were used for PCR reactions using primers and conditions described below. The complete removal of DNA in RNA samples was confirmed by no amplification of these PCR reactions. RNA was then quantified by absorbance at 260 nm using a Nanodrop spectrophotometer. cDNAwas synthesized from 1 μg of RNA using SuperScipt™ III reverse transcriptase (Invitrogen, CA,
Organic anions in exudates and microbial communities were compared between the soil categories (rhizosphere vs non-rhizosphere) and tree lines (GM vs control pines) at corresponding depths. Because of the significant difference between rhizosphere and non-rhizosphere soils in both organic anion profiles and microbial DGGE patterns, tree line treatment analysis was carried out separately on rhizosphere and non-rhizosphere soils. The detection frequencies of OAs between samples collected using different sampling techniques or extraction methods were analysed using Chi-square test for independence and Yates correction for continuity, with the hypothesis that the detection frequency of each OA is independent of sampling and extraction methods. The concentrations of individual organic anion components and total C were log10 transformed and analysed using ANOVA according to the treatment effects (soil categories, tree lines). In samples where no OAs were detected, the values were set to half the limit of detection of the
Exudate extraction and analysis Water-soluble exudate extraction Water-soluble root exudates were extracted from the rhizosphere and non-rhizosphere soil samples collected by in situ or destructive samplings using a method modified from Schefe et al. (2008). Briefly, sterile deionised water was added to each root-rhizosphere or non-rhizosphere sample at a ratio of 5 ml to 1 g sample. The samples were then shaken for 10 min at 100 rpm, followed by centrifugation at 2,800g for 10 min. The supernatant containing water-soluble components was stored at −20°C until analysis by HPLC. Exudate analysis
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HPLC method (Shi et al. 2011a). Chi-square was analysed using software R and the ANOVA analyses were conducted using Genstat™ 11 (VSN International Ltd, UK). Similarity matrices derived from analysis of DGGE profiles were reduced to five dimensions using principle coordinate analysis, and linear discriminant analysis was used to evaluate the differences between the treatments. The 95% confidence regions around the group means were produced by the GenStat DISCRIMINATE procedure (Payne et al. 2007). The significance of the treatment differences were assessed using a Hotelling T2-test (Hotelling 1947).
Results Organic anions in root exudates and soil solutions Detection All 12 targeted OAs were detected in rhizosphere samples (Table 1), although detection of each anion varied between sampling techniques and extraction methods. With the exception of succinate, all OAs were detected in the rhizosphere samples collected in situ by AEM (Table 1). Formate, lactate, acetate,
shikimate and fumarate were the most frequently detected OAs (>90%). While succinate was not detected by AEM, it was found in 15 out of 32 water-soluble root exudate samples collected by in situ sampling. In the water-soluble extractions of rhizosphere samples collected with in situ sampling, shikimate, fumarate, malate, quinate, citrate and malonate were detected at high frequencies (≥ 97% of samples), while maleate was not detected in any sample despite being detected by AEM at a frequency of 84% (Table 1). The detection frequencies of eight out of 12 targeted OAs in rhizosphere exudate samples were significantly linked (P
