To culture or not to culture: a snapshot of culture-dependent and culture-independent bacterial diversity from peanut rhizosphere

Abstract

Background Sequencing driven metagenomics studies have been instrumental in various aspects of microbiology including identification of newer taxa. While this culture-independent approach has its own merits and demerits, several studies have focussed on comparing it with traditional culture-dependent (CD) approach. However, most of these comparative studies rely on Sanger sequencing of complete 16S rRNA gene from pure culture colonies to determine the culturable bacterial diversity. This approach undercounts culturable diversity as only fewer isolates are selected, sequenced, and identified. Methods In this study, we have used an Illumina based partial 16S sequencing to identify all the microbes growing on the media and directly comparing with its culture-independent (CI) counterpart. Eight different media were used to target different organisms from soil. Diversity on these media were compared with their CI counterpart. The NGS data was analysed using DADA2 to provide more resolution to the data. Results In line with studies of similar nature, current study presented higher bacterial diversity in CI approach. However, the current study reflected that a greater number of sequence variants were missed out in CI approach as compared to number of sequence variants shared with CD approach. We observed around 322 (5.98%) ASVs (Amplicon Sequence Variants) exclusively present in CD samples while, 234 (4.35%) ASVs were shared between both approaches. Most of these 322 CD exclusive ASVs were classified as Enterobacteriaceae family and Bacillus genus, with several ASVs annotated at the species level as well, and these organisms are more commonly observed in soil and were also detected in CI approach. Furthermore, 22 genera were exclusively detected in CD samples, most of which were reported from soil and water.

Metagenomic evaluation of peanut rhizosphere microbiome from the farms of Saurashtra regions of Gujarat, India

“…This might be due to the very low abundance of those organisms, which is a limitation of CI approach. Comparing all the genera of both the approaches, revealed that total 38 genera were exclusively found in CD samples, that is similar to the studied by Hinsu et al 57 . Most of these genera are commonly found in soil, forest and water sources including marine water.…”

“…Furthermore, for comparative study, partial 16S rRNA gene was sequenced, similar to metagenomics, rather than sequencing the entire 16S rRNA gene, through Sanger sequencing as done in all previous researches 32 – 34 . As per our knowledge, a similar approach has also been successfully applied by Zehavi et al for the study of ruminal microbiota and in our previous study on rhizosphere microbiome 56 , 57 . This approach could be helpful to analyze the presence-absence based study of microbial diversity.…”

Microbial Profiling of Biltong Processing Using Culture-Dependent and Culture-Independent Microbiome Analysis

“…One reason could be the recovery medium used for culture-dependent isolation was unsupportive of Lactococcus spp. Some investigators have examined as many as four [62] to eight [63] growth media for comparison with culture-independent methods for improved detection of OTUs. Anguita-Maeso et al (2020) examined xylem-colonizing bacteria of olive plants and found only 41% of the total genera using culture-dependent methods as was found by culture-independent methods by Illumina MiSeq 16S rRNA sequencing using NGS [62].…”

Yeast metagenomics: analytical challenges in the analysis of the eukaryotic microbiome

“…However, these techniques have well-known limitations. For instance, many species remain undetected because appropriate culture conditions are either unknown or challenging to reproduce [ 41 ] . Moreover, culture methods are time-consuming and hardly suitable for high-throughput analysis.…”