## 2 Soil-Borne Plant Pathogens and Microbiota Determine Disease Suppression
### Omics Approach for Studying Soil Microbiome
Nowadays, the culture-independent strategy is increasingly used. The development of NGS allows to perform metabacoding analyses involving the amplification and sequencing of specific marker genes to identify a whole community in a DNA sample without the need of cloning or separation steps. Finally, NGS involving the random sequencing of the fragmented DNA extract (shotgun metagenomics approach) and allowing the study of the microbial diversity and the prediction of associated gene functions, was used to perform metagenomics soil analyses. It is important to underline that each approach displays its own strengths and weaknesses [29]. On the one hand, the culture-dependent strategy allows isolating the microorganisms and further characterization of their biochemical and functional traits. However, this way is very laborious, time-consuming, and has a limited capacity to cover the whole diversity of microorganisms because it is dependent on many parameters, such as the culture media employed. Indeed, the concept of \"un-culturable microorganism\" was highlighted for the first time in the early 20 th century, where there were far fewer colonies able to grow on the medium than the number of cells observed by microscopy [176; 177]. Nevertheless, this limit can now be bypassed with the use of various culture media leading to the development of the \"culturomics\" approach. On the other hand, the culture-independent strategy is more labor/cost-effective in studying the abundance, richness and diversity of the microorganisms' community, as well as in identifying the uncultivable ones. This strategy can highlight the relative abundance of the observed OTUs in metabarcoding and the potential functions of associated genes in metagenomics study. Despite a bias that can be introduced by the DNA extraction step when studying the microbial relative abundance, soil amendment with an exogenous microbial community and the improvement of the DNA extraction protocols can help to standardize the results [41]. Another constraint is the difficulty to reach the lowest taxonomic levels due to the limited amplicon length when using the first-generation sequencers (Roche 454-pyrosequencer), which was discontinued in 2016 [29; 178; 179]. Indeed, most of the metabarcoding studies related to soil microbiome diversity in coffee plantations under organic and conventional production in tropical agro-ecosystems were performed with the second-generation sequencers (Illumina HiSeq and Illumina HiSeq) that allowed sequencing of the hypervariable regions V3-V4 of bigger markers such as the 165 rDNA gene for bacteria and prokary, and the 188 rDNA gene for eukary, or smaller markers such as the ITS gene region for fungi,omycetes and yeasts [180; 181]. Moreover, it has already been demonstrated that smaller sequences do not achieve the highest taxonomic resolution levels for fungi, which are obtained instead with the full length 165 rDNA for bacteria [178]. By contrast, the latest technologies of third (Ion Torrent) and fourth generations (PacBio and Oxford Nanopore) allowed to generate longer sequences if compared to the others' NGS, but this is done at the expense of the quality due to higher sequencing error rate [29]. However, the full capacity of the platforms remained unexploited as they sequenced only the ITS1-5.85-ITS2 region of rDNA (<1 kb). The latest technologies of the third generation allow overcoming this problem by generating longer high-quality sequences if compared to the advent of the high-throughput sequencing [178]. Finally, it is important to remind that the data generated by Illumina and Ion Torrent need laborious and advanced statistical analyses for sequence processing by using algorithms implemented by bioinformatics analyses that still need to be improved [182; 183; 184]. In concluding this issue, it must be recalled that the phylogenetic characterization of microbiota based on DNA analyses does not reflect the real biological activity of the microbial community.