A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments

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Abstract

A major obstacle in the culture-independent estimation of the abundance of bacteria associated with plants is contamination with plant organelles, which precludes the use of universal rRNA bacterial primers in quantitative PCR applications. We present here a PCR-based method that allows a priori determination of the degree of chloroplast and mitochondrial contamination in DNA samples from plant environments. It is based on differential digestibility of chloroplast, mitochondrial and bacterial small subunit rRNA gene amplicons with the restriction enzymes AfeI and BbvCI. Using this method, we demonstrated for field-grown lettuce plants that even a gentle washing protocol, designed to recover the microbial community and its metagenome from the leaf surface, resulted in substantial contamination with chloroplast DNA. This finding cautions against the use of universal primer pairs that do not exclude chloroplast DNA from amplification, because they risk overestimation of bacterial population sizes. In contrast, contamination with mitochondrial 18S rRNA was minor in the lettuce phyllosphere. These findings were confirmed by real-time PCR using primer sets specific for small subunit rRNA genes from bacteria, chloroplasts, and mitochondria. Based on these results, we propose two primer pairs (534f/783r and mito1345f/mito1430r) which between them offer an indirect means of faithfully estimating bacterial abundances on plants, by deduction of the mito1345f/mito1430r-based mitochondrial count from that obtained with 534f/783r, which amplifies both bacterial and mitochondrial DNA but excludes chloroplast. In this manner, we estimated the number of total bacteria on most leaves of field-grown lettuce to be between 105 and 106 g 1 of leaf, which was 1–3 orders of magnitudes higher than the number of colony-forming units that were retrieved from the same leaf surfaces on agar plates.

Introduction

In recent years, there has been a dramatic increase in the number of culture-independent approaches to the study of plant-associated bacterial communities (Chelius and Triplett, 2001, Yang et al., 2001, Lambais et al., 2006, Jackson et al., 2006, Sun et al., 2008, Redford and Fierer, 2009, Redford et al., 2010). Irrespective of the method employed to isolate DNA from these communities, the extracted microbial DNA is generally contaminated with DNA of plant origin, in particular chloroplasts and mitochondria (Chelius and Triplett, 2001, Sakai et al., 2004, Sun et al., 2008). Since the chloroplast 16S and mitochondrial 18S rRNA genes share high sequence similarity with bacterial 16S rRNA sequences (Sakai et al., 2004), contamination with plant DNA poses a serious challenge for the application of PCR-based methods to profile and quantify bacterial populations in plant environments.

Several primer pairs have been reported which amplify bacterial 16S rRNA genes to the exclusion of chloroplast sequences (Chelius and Triplett, 2001, Sakai et al., 2004, Edwards et al., 2007). One of these, 799f/1492r, was originally used to analyze bacterial endophytes of maize roots (Chelius and Triplett, 2001). While primer 1492r (Lane 1991) is considered universal (i.e. it targets bacterial, chloroplast, as well as mitochondrial rRNA genes), 799f features several mismatches with chloroplast 16S rRNA gene sequences that prevent the latter from being amplified. In the original paper, evidence for this exclusion was the absence of chloroplast DNA sequences in a clone library that was derived from non-sterile maize roots using the 799f/1492r pair (Chelius and Triplett, 2001). This primer set has since been used widely for the construction of 16S rRNA gene clone libraries from below- and above-ground plant environments (Kadivar and Stapleton, 2003, Sun et al., 2008, Sagaram et al., 2009, Redford and Fierer, 2009).

Sakai et al. (2004) reported an alternative to 799f which allows it to be used as a reverse primer. Primer mix 783r-abc consists of three primers that are reverse complementary to 799f, have several nucleotides added or removed at the 5′ and 3′ end, and between them offer a slightly broader target range than 799f by increased nucleotide ambiguity. Primer mix 783r-abc was used together with universal primer 63f (Sakai et al., 2004) for the study of bacterial communities of wheat and spinach roots by terminal restriction fragment length polymorphism (T-RFLP) analysis. No fragments representing amplified chloroplast sequences were found in the T-RFLP profiles of these communities (Sakai et al., 2004), which offers convincing evidence for the exclusive nature of 799f also in its reverse orientation.

Primer pairs 799f/1492r and 63f/783r-abc are not recommended for the culture-independent, PCR-based quantification of total abundances of bacteria associated with plants. The sizes of their respective amplicons exceed what is considered optimal for real-time PCR purposes (Smith and Osborn 2009). Edwards et al. (2007) conceived of producing an amplicon with a smaller size, by pairing 799R2, a reverse derivative of 799f having its degenerate bases removed, with 520f, which is a truncated derivative of universal primer 518r (Muyzer et al., 1993). This primer pair was successfully used to quantify by real-time PCR bacterial population sizes on perennial ryegrass (Edwards et al., 2007).

One feature that bacterial primers 779R2, 799f and 783r-abc share with each other is that they are not exclusive of mitochondrial 18S rRNA gene sequences. For the construction of clone libraries involving primer pairs such as 799f/1492r, this is not an issue, because the mitochondrial product with these primers is larger than that of bacteria, which allows exclusion of the former by size-separation on an agarose gel (Chelius and Triplett, 2001). In T-RFLP analyses using primer pairs such as 63f/783r-abc, mitochondrial DNA typically renders a specific peak that can be similarly excluded from the analysis (Sakai et al., 2004). However, in quantitative, real-time PCR, mitochondrial amplicons cannot be distinguished from their bacterial counterparts by size or other properties, and depending on the contamination with mitochondrial DNA, this may lead, just as is true for primers that do not exclude chloroplasts, to overestimation of bacterial abundances by real-time PCR.

Methods that are specifically designed for the qualitative and quantitative estimation of mitochondrial and chloroplast contamination in DNA preparations from plant-associated bacterial communities are currently not available, yet would be very useful for assessing the risk of overestimating bacterial population sizes due to contaminating plant organelle DNA. Here, we describe a PCR-based toolbox that allows a faithful estimation of the degree of chloroplast and mitochondrial contamination in DNA isolated from plant microbiota. Part of the toolbox involves the exploitation of restriction enzymes that uniquely recognize chloroplast and mitochondrial ribosomal RNA gene amplicons. The toolbox also offers a novel set of primer pairs for the accurate estimation of bacterial abundances in plant environments, by using chloroplast-exclusive primers and by subtracting mitochondrial estimates.

Section snippets

DNA extraction from phyllosphere microbial communities, Escherichia coli, and aseptically grown lettuce seedlings

Field-grown Romaine lettuce (Lactuca sativa) plants were harvested from production regions in Imperial (CA) and Yuma (AZ) at different times during the 2009/2010 growing season. Per field, two samples were taken just prior to harvest. Each sample contained four lettuce heads, and from each lettuce head, two outer most and two inner leaves from the fourth leaf circle were picked. Microorganisms were recovered from leaf surfaces as described by Redford and Fierer (2009). Briefly, the 16 leaves

Assessment of plant DNA contamination by digestion of SSU rRNA amplicons

Analysis of the 16S rRNA gene sequence from lettuce (Lactuca sativa) chloroplasts (accession number NC_007578) revealed a recognition site for the restriction enzyme BbvCI (5′-GCTGAGG-3′) which was not shared with the mitochondrial sequence (Fig. 1A). Conversely, the lettuce mitochondrial 18S rRNA gene featured a recognition site for AfeI (5′-AGCGCT-3′) which was absent from the chloroplast sequence (Fig. 1A). We exploited this difference to estimate plant organelle contamination in DNA

Discussion

We presented several methods that should be of interest to those who study plant-associated bacterial communities in a culture-independent, PCR-based manner. The first allows a quick assessment of the degree to which samples are contaminated with chloroplast DNA, which is a common problem in these types of study. The method is based on the BbvCI digestion of PCR products resulting from amplification with the commonly used universal bacterial primers 27f-YM and Eub518r. We demonstrated proof of

Acknowledgements

This project was supported by the California Leafy Greens Research Program and Center for Produce Safety (contract number LGR-2009-20). We acknowledge Dr. Trevor Suslow and Adrian Sbodio for help with providing lettuce samples, and Dr. Maria Marco for valuable feedback on an earlier draft of the manuscript. We also thank three anonymous reviewers whose comments were instrumental in improving the quality of our manuscript.

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