Tracking Fungal Community Responses to Maize Plants by DNA- and RNA-Based Pyrosequencing

Eiko E Kuramae,Mattias De Hollander,Mari Moora,Remy Hillekens,Marcel G A Van Der Heijden,Erik Verbruggen,Wilfred F M Röling,George A Kowalchuk

doi:10.1371/journal.pone.0069973

Abstract

We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.

Highlights

Numerous soil processes are primarily, and some even exclusively, carried out by soil fungi
We examined the potential impact of four maize plant cultivars, consisting of two genetically modified cultivars (GM) and two near-isogenic non-Genetic Modification (GM) cultivars, on soil-borne fungal communities in a pot-based experiment
The use of a more variable marker, such as the Internal Transcribed Spacer (ITS) regions [33], which can discriminate to the subspecies level, would no doubt yield larger OTU numbers than we recovered

Summary

Introduction

Numerous soil processes are primarily, and some even exclusively, carried out by soil fungi. The fungi responsible for these key soil functions are in direct contact with plants and plant materials. They may be especially vulnerable to alterations in e.g. plant defenses and carbohydrate composition and availability [3,4], as generated by Genetic Modification (GM) or other plant variety specific differences. High-throughput sequencing approaches, such as 454 pyrosequencing, have greatly expanded the power of such nucleic acid-based assessments of complex fungal communities [6,7]. There is currently no consensus on whether DNA, RNA or both should be targeted to obtain the most meaningful assessment, and how communities obtained by analysing either nucleic acid type relate to each other

Methods

Results

Conclusion