Abstract:
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 contentmust 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 sorghumendophytic
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.
