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

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.

Arbuscular mycorrhizal fungal diversity in rhizosphere spores versus roots of an endangered endemic tree from Argentina: Is fungal diversity similar among forest disturbance types?

“…Third, AMF root-colonizing taxa could vary along the target plant life cycle (Kuramae et al, 2013). For instance, Husband et al (2002) showed that the richness and diversity of AMF community colonizing Tetragastris panamensis roots decreased with seedlings age.…”

Differential responses of soil bacteria, fungi, archaea and protists to plant species richness and plant functional group identity

“…The PCR amplification of 16S had an initial annealing temperature of 95°C (5 min), then 30 cycles with denaturation at 95°C (30 s), annealing at 53°C (1 min), extension at 72°C (1 min) and final extension 72°C (10 min). We amplified 18S rRNA gene fragments from fungi and protists with primer FR 1 and the modified version of FF390 5′‐CGWTAACGAACGAGACCT‐3′ designed to also include the Glomeromycota (Kuramae et al., ). The amplification mixture contained 0.1 pmol/μl of each primer, 1 μl DNA, 200 μ m dNTPs, 0.056 U FastStart™ High Fidelity PCR System (ROCHE), 10× PCR buffer, 2.5 μ m MgCl 2 and 0.5 mg/ml bovine serum albumin.…”

“…The PCR amplification of 16S had an initial annealing temperature of 95°C (5 min), then 30 cycles with denaturation at 95°C (30 s), annealing at 53°C (1 min), extension at 72°C (1 min) and final extension 72°C (10 min). We amplified 18S rRNA gene fragments from fungi and protists with primer FR 1 and the modified version of FF390 5 0 -CGWTAACGAACGAGACCT-3 0 designed to also include the Glomeromycota (Kuramae et al, 2013).…”

Biogeography of aquatic hyphomycetes: Current knowledge and future perspectives

“…Applying Next Generation Sequencing (NGS) to cDNA (the synthesis of cDNA from RNA is only possible in metabolically active species) (e.g. Kuramae et al, 2013) will provide greater insights into metabolically active species.…”