Bacterial community in the rhizosphere of the cactus species Mammillaria carnea during dry and rainy seasons assessed by deep sequencing G. Torres-Cortés, V. Millán, A. J. Fernández-González, J. F. AguirreGarrido, H. C. Ramírez-Saad, M. Fernández-López, et al. Plant and Soil An International Journal on Plant-Soil Relationships ISSN 0032-079X Volume 357 Combined 1-2 Plant Soil (2012) 357:275-288 DOI 10.1007/s11104-012-1152-4
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Author's personal copy Plant Soil (2012) 357:275–288 DOI 10.1007/s11104-012-1152-4
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Bacterial community in the rhizosphere of the cactus species Mammillaria carnea during dry and rainy seasons assessed by deep sequencing G. Torres-Cortés & V. Millán & A. J. Fernández-González & J. F. Aguirre-Garrido & H. C. Ramírez-Saad & M. Fernández-López & N. Toro & F. Martínez-Abarca
Received: 22 November 2011 / Accepted: 26 January 2012 / Published online: 3 March 2012 # Springer Science+Business Media B.V. 2012
Abstract Background and aims The Tehuacán-Cuitcatlán reserve is an area of unique plant biodiversity mostly in the form of xerophytes, with exceptionally high numbers of rare and endemic species. This endemism results partly from the characteristics of the climate of this area, with two
Responsible Editor: Petra Marschner. Electronic supplementary material The online version of this article (doi:10.1007/s11104-012-1152-4) contains supplementary material, which is available to authorized users. G. Torres-Cortés : V. Millán : A. J. Fernández-González : M. Fernández-López : N. Toro : F. Martínez-Abarca (*) Departamento de Microbiología y Sistemas Simbióticos, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda 1, 18008 Granada, Spain e-mail:
[email protected] J. F. Aguirre-Garrido : H. C. Ramírez-Saad Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Xochimilco, 04960 México, DF, Mexico Present Address: G. Torres-Cortés Genetics, University of Munich (LMU), 82152 Martinsried, Germany
distinct seasons: rainy and dry seasons. Although rhizosphere communities must be critical in the function of this ecosystem, understanding the structure of these communities is currently limited. This is the first molecular study of the microbial diversity present in the rhizosphere of Mamillaria carnea. Methods Total DNA was obtained from soil and rhizosphere samples at three locations in the Tehuacán Cuicatlán Reserve, during dry and rainy seasons. Temperature gradient gel electrophoresisis (TGGE) fingerprinting, 16S rRNA gene libraries and pyrosequencing were used to investigate bacterial diversity in the rhizosphere of Mammillaria carnea and changes in the microbial community between seasons. Results Deep sequencing data reveal a higher level of biodiversity in the dry season. Statistical analyses based on these data indicates that the composition of the bacterial community differed between both seasons affecting to members of the phyla Acidobacteria, Cyanobacteria, Gemmatimonadetes, Plantomycetes, Actinobacteria and Firmicutes. In addition, the depth of sequencing performed (>24,000 reads) enables detection of changes in the relative abundance of lower bacterial taxa (novel bacterial phylotypes) indicative of the increase of specific bacterial populations due to the season. Conclusions This study states the basis of the bacterial diversity in the rhizosphere of cacti in semi-arid environments and it is a sequence-based demonstration of community shifts in different seasons.
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Keywords Microbial community . Soil bacteria . 16S rRNA analysis . Mammillaria carnea . Tehuacán Cuicatlán reserve . 454-pyrosequencing
Introduction The Tehuacán-Cuicatlán Valley is located in the states of Puebla and Oaxaca (Mexico), and covers an area of about 10,000 km2 (Fig. 1). The valley presents a complex topography, extending from elevations of 500 to 3,200 m. It is considered an arid area with a semiarid climate. Indeed, there are two main seasons: dry (from mid-October to April) and rainy (from May to midOctober; Enge and Whiteford 1989). In general, soils are rocky and shallow, no more than 15 cm depth with high content of calcium and organic matter. This last ranges from 2% in the slopes of the valley to 5% at the top and bottom of the hills. Soil classes are calcaric fluvisol and calcaric regosol with sandy loam texture and pH ranging between 7 and 8.6 (Rivera-Aguilar et al. 2009 and references in). The flora has neotropical affinities, with almost 2,700 species present, 11% of which
Fig. 1 Geographic location of the sampling sites. a Location of the biosphere reserve in Mexico. b Tehuacán Cuicatlán reserve is located between states of Puebla and Oaxaca. c Sampling sites are indicated with arrows: VT1 and VT2 correspond to sites close to Valerio Trujano village and are closer to each other than to the To (Tomellin) sampling site
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are endemic to the valley (Smith 1965; Dávila et al. 2002). This endemism is particularly remarkable in the cactacea genus Mammillaria. It is known that plants have a profound effect on the abundance, diversity and activity of soil microorganisms living in close proximity with their roots - a soil zone defined as rhizosphere (Berg and Smalla 2009). There is little knowledge on rhizosphere microbiology of desert plants, although the rhizosphere effect is qualitatively and quantitatively more pronounced in desert soils as compared to soils in a humid climate (Chanal et al. 2006; Fierer et al. 2007; Bachar et al. 2010). On the other hand, studies on the responses of organism communities to precipitation have led to searches for patterns of diversity across a wide spectrum of taxonomic groups (Hawkins et al. 2003). Usually, an increase in plants and animals diversity is observed with increasing water availability. However, very little is known about changes in microbial diversity (Bachar et al. 2010; Angel et al. 2010). Various large-scale surveys conducted in recent years have shown that different ecosystems support unique microbial populations and indicated that microbial assemblages may have small(within-site) and large-scale biogeographic distributions (Zhou et al. 2002; Green and Bohannan 2006; Fierer and Jackson 2006; Bahl et al. 2011). Comparative analysis of 16S rRNA gene sequences is an excellent method for a first phylogenetic afilliation of well characterized, but also potentially new and poorly classified organisms (Rosselló-Mora and Aman 2001). Most approaches generated 16S rRNA gene amplicons and analyzed them through the sequencing of clone libraries (Chow et al. 2002; He et al. 2006), by denaturing or temperature gradient gel electrophoresis (DGGE; TGGE) (Muyzer et al. 1993; Villadas et al. 2007), or by terminal restriction fragment length polymorphism (T-RFLP; Fierer and Jackson 2006). These approaches can reveal differences in community structure, but are of limited use for the assessment of diversity (Osborn et al. 2000; Hartmann and Widmer 2006). Phylogenetic groups present at low abundance potentially playing key roles in functioning ecosystem are not assessed (Schloss and Handelsman 2006; Elshahed et al. 2008). It has also been shown that several thousand clones are required from complex bacterial communities for robust measurements and estimates of community diversity parameters (Morales et al. 2009). Large-scale pyrosequencing of partial 16S rRNA genes have recently provided comprehensive insight into the biogeography
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of bacterial soil communities, which appear to be extremely diverse, demonstrating that it is difficult to obtain deep coverage by cloning strategies (Roesch et al. 2007; Kolton et al. 2007; Nacke et al. 2011; Köberl et al. 2011). In this study, we characterize the bacterial diversity associated with samples of the rhizosphere of Mammilaria carnea plants by analyzing amplicons of the 16S rRNA gene and we investigated the effect of natural water availability on the pattern of microbial diversity in this semi-arid ecosystem. As suggested in other studies (Bachar et al. 2010), we hypothesize that water availability shapes rhizosphere bacterial communities and results in different diversity patterns during dry and rainy seasons.
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The pH was determined in 1:2 w/v suspensions of soil to deionized water. DNA extraction DNA was extracted in the week following sample collection from 500 mg of soil per sample. Total DNA was extracted from soils with a CTAB-based extraction protocol, as described elsewhere (Porteous et al. 1994). The extracted DNA was cleaned, concentrated on AMICON YM100 filters (Millipore, Billerica, MA, USA), according to the manufacturer’s protocol and quantified with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The integrity of the DNA extracted from the soil was confirmed by agarose gel electrophoresis.
Materials and methods TGGE Site description and sample collection The study has been carried out at the Tehuacán-Cuicatlán biosphere reserve in central-south Mexico (Fig. 1). The annual mean temperature in the reserve is 24.5°C. Mean annual precipitation ranges from 250 mm to 500 mm, falling from May through October, with main rainfall levels from June to September (Enge and Whiteford 1989). We selected three sampling sites, well preserved and with little human impact: two located close together, near to the village of Valerio Trujano (VT1 and VT2) and a third site close to Tomellin village (To). Samplings were performed in August, at the end of the rainy season, and in March during the dry season (Supplementary Fig. S1). At each sampling site and season, the soil associated to roots of six plants of the cactus species M. carnea were pooled and homogenized to one composite sample of 100–200 g, respectively. Coarse roots and particles (>2 mm) were removed and soil samples were stored at 4°C. Bulk soil was collected in the vicinity of the chosen cacti at each sampling site, but considering spots without plants. Determination of soil water content, nitrate and pH Soil water content was determined by gravimetric analysis; 10 g of each soil sample were dried overnight at 120 ° C. Water content was calculated as the difference between humid and dry weight. The amount of nitrate in the soil samples was calculated using the colorimetric reaction protocol described by Kempers (1974).
DNA from all composite soil samples (10 ng) was used as the template for a nested PCR targeting the 16S rRNA gene of α-Proteobacteria (primers F203α/L; Villadas et al. 2007); a second PCR was performed with primers F984GC/R1378. Primer sequences and conditions for PCR and TGGE have been described elsewhere (van Dillewijn et al. 2002). We used the Maxi TGGE system, according to the manufacturer’s instructions (WhatmanBiometra, Göttingen, Germany) to resolve the fingerprints. TGGE profiles were analyzed with the Quantity One ® 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA). Similarities between the patterns (70% are shown. Genera, species, or alternative identification in database are followed by their GenBank accession numbers. Most of the Gp4 clusters described in Table 3 formed branches that were grouped to improve the visibility of the tree (the numbers of sequences found in rainy (R) and dry (D) season samples are indicated in parentheses). Sequences (and triangles) are shown in gray and black for dry and rainy season-specific clusters, respectively. The 16SrRNA sequences of members of Acidobacteria class Gp2 were used as outgroup (Accession nos: AJ519364, EF019077, AY913350, EF018507). Scale bar00.1 changes/site
E classified within family XIII cyanobacteria containing a dominant cosmopolitan cyanobacterium, Microcoleus chthonoplastes, which occurs in hypersaline microbial mats (Green et al. 2008). The remaining cyanobacterial clusters form a branch related to Pseudanabaenaceae cyanobacterium (EF654061). Among them, the cluster A stands out because it contains 91.30% of the cyanobacterial sequences of the rainy season sample. Cluster A branches off within the lineage of the Streptophyta group, and its 16S rRNA gene sequences were found to be related to those of uncultured bacteria and/or eukaryotic plastids (Fig. 4).
Discussion In this study, we described the bacterial community associated with the M. carnea rhizosphere in two
different seasons. We characterized their relative abundance, diversity and composition based on 16SrRNA gene profiles obtained by TGGE; and sequence data obtained by the sequencing of a library of clones and pyrosequencing. Richness index values suggested that the microbial community was significantly affected by the season. In addition, plant species are known to shape the rhizosphere microbial community, possibly through the composition of their root exudates (Somers et al. 2004). Our work partially confirmed this assumption; at least in the rainy season, a fingerprint based on α-proteobacterial subpopulation was associated with M. carnea plants, with independence of the three sampling sites selected. The 16S rRNA amplicons obtained in this study revealed that the diversity of the M. carnea rhizosphere, at the phylum level, was greater than reported for previous similar soil datasets from arid areas (Saul-
Author's personal copy 284 Fig. 4 Neighbor-joining phylogenetic tree of partial 16SrRNA gene reads for cyanobacteria described in Table 3. Tree topology is shown as indicated in Fig. 3. The 16SrRNA sequences of Brochothrix campestris, B. thermosphacta and an uncultured bacterium (Accession nos.: AY543038; AY543028; AY796043, respectively) from the phylum Firmicutes were used as outgroup
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Tcherkas and Steinberger 2011). The particular high level of diversity found in the dry season contrasts with other studies reporting low levels of fluctuation of bacterial richness between seasons in soils with different water availabilities (Warren-Rhodes et al. 2006; Bachar et al. 2010). The most abundant phylum in this rhizosphere was Acidobacteria. This result is in agreement with its predominance of this phylum in semi-arid soil environments (Bachar et al. 2010), and also in other mature soils (Tarlera et al. 2008). This abundance can be probably due to the oligotrophic nature described for these bacteria (Fierer et al. 2007). Its relative abundance increased during the rainy season, but their diversity decreased. This increment contrast with results reported for soils where Acidobacteria is not the predominant phylum that its relative abundance decreases with increasing water availability (Castro et al. 2010). At the subphylum level the predominance of Class Gp4 and, to a lesser extent, Gp3, Gp6 and Gp7 is consistent with the findings of Jones et al. (2009) that the abundance of some subgroups of Acidobacteria (Gp1, 2, 3, 12 and 13) is negatively correlated with pH whereas that of other groups, such as the predominant classes found here, is positively correlated with pH. Phylum Actinobacteria is the second most abundant group in the M. carnea rhizosphere. Within this phylum, about 80% of the sequences obtained belonged to the class Actinobacteria (order Rubrobacteriales and subclass Actinobacteridae). Members of the family Rubrobacteriaceae seem to be specific to semi-arid environments (Bachar et al. 2010). Proteobacteria sequences were also highly abundant, accounting for 13% to almost 18% of all sequences found in this study, but this phylum did not predominate as it does in other soil samples (Tamames et al. 2010). The phylum Firmicutes increased in abundance during the dry season, mostly due to bacteria from the class Clostridia which contains spore-forming bacteria to ensure their survival through periods of environmental stress, such as low water availability (Tamames et al. 2010). Bacteria favoured in rainy season samples could be an indication of r-selected life-history strategy, typified by rapid responses to high resource availabilities. In contrast, abundant groups in the dry season perhaps reflect a slow growing K-selected strategy suited by low resource availability (Fierer et al. 2007; Cruz-Martínez et al. 2009). It has been described that Bacteria belonging to the Acidobacteria phylum were most abundant in soils
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with very low resource availability (Fierer et al. 2007); but there is an enormous amount of phylogenetic and physiological diversity within a targeted phylum and it is unlikely that an entire phylum would share common ecological characteristics. By examining group abundances at finer levels of taxonomic resolution (Table 3 and Figs. 3 and 4), ecological divisions (dry and rainy seasons) may become more apparent and subsets of each group could be classified as characteristic of particular environmental conditions. Thus, under 97% 16S rRNA gene sequence similarity we found 24 clusters, with significant differences (p-value
