Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

Abstract

Keywords:Metagenomic DNA extraction Endophytic bacteria Sorghum root and stem t-RFLP Pyrosequencing Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB-and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDSbased DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities.

Bacterial endophyte communities of three agricultural important grass species differ in their response towards management regimes

“…The genera Massilia, Pseudomonas , and Limnohabitans were predominant in the three grass species. This is only partly in line with a previous study showing that Pseudomonas, Rhizobium , and Bacillus were the most abundant genera in roots and shoots of sorghum21. Similar results were obtained by Robinson et al 7.…”

“…In total, 71 samples of the grass species L. perenne, D. glomerata , and F. rubra in two consecutive years were analyzed. Consistent with previous work72021, bacterial endophyte communities were dominated by five phyla. The genera Massilia, Pseudomonas , and Limnohabitans were predominant in the three grass species.…”

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…Extraction requirements and recommendations A range of methods are used to extract DNA from plant tissues, including fungal and bacterial endophytes Maropola et al 2015). Recent extractions of arbuscular mycorrhizal fungal (AMF) DNA from plant roots have used the DNeasy PowerSoil ® kit Padamsee et al 2016), or the DNeasy Plant kit for AMF in the roots of Ammophila arenaria (Johansen et al 2015).…”

“…It is possible that the DNeasy PowerSoil ® kit gives superior results in samples of tree roots that are higher in phenolic compounds in comparison to softer tissue like grass roots. DNeasy PowerSoil ® or PowerMax ® kits are routinely used for sample material, including plant tissues, that are high in inhibitory compounds (Dineen et al 2010;Maropola et al 2015), even though relatively low DNA yields are reported for some sample material (Maropola et al 2015). While there is little evidence upon which to base favouring one extraction method over others, standardisation on one method shown to be adequate (or at least not inadequate) will improve the comparability of results obtained from different laboratories.…”

Bacterial endophytes in agricultural crops and their role in stress tolerance: a review

“…Tsurumaru et al (2015) analyzed a metagenome of the bacterial community associated with the taproot of sugar beet (Beta vulgari L.) The study found that Alphaproteobacteria are dominant, followed by the Actinobacteria and the Betaproteobacteria. Another metagenomic study of the sorghum root and stem microbiome revealed that the two tissues harboured significantly different composition of bacterial communities, but both were dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas (Maropola et al, 2015).…”