Metagenomics Next Generation Sequencing (mNGS): An Exciting Tool for Early and Accurate Diagnostic of Fungal Pathogens in Plants

Abstract

Crop output is directly impacted by infections, with fungi as the major plant pathogens, making accurate diagnosis of these threats crucial. Developing technology and multidisciplinary approaches are turning to genomic analyses in addition to traditional culture methods in diagnostics of fungal plant pathogens. The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping identification of organisms, identification at the species level, illumination of metabolic pathways, and determination of microbiota. Moreover, the data obtained so far show that this new approach is promising as an emerging new trend in fungal disease detection. Another approach covered by mNGS technologies, known as metabarcoding, enables use of specific markers specific to a genetic region and allows for genotypic identification by facilitating the sequencing of certain regions. Although the core concept of mNGS remains constant across applications, the specific sequencing methods and bioinformatics tools used to analyze the data differ. In this review, we focus on how mNGS technology, including metabarcoding, is applied for detecting fungal pathogens and its promising developments for the future.

Keywords: ITS; bioinformatic analysis; fungal pathogens; mNGS; metabarcoding.

Figure 1

Applications of mNGS technology in different fields.

Figure 2

mNGS and metabarcoding workflow chart for the sample obtained from the infected leaf. The workflow highlighted in red shows metabarcoding pathways, which use specific metabarcodes for fungal detection, and the “black” arrow shows the mNGS pathways. In the workflows, the PCR stage is optional, and after sequencing and bioinformatic analysis, metabarcoding shows genotyping identification. mNGS indicates fungal species identification, microbial diversity, pathway detection, and genotyping identification. Both techniques seem to include the same steps; however, the algorithms (the bioinformatics analysis) differ. In metabarcoding, certain parts of the genome are sequenced using target-specific barcodes, and in mNGS, either a partial or a whole genome is sequenced by reference-based comparison with the prepared library. Both approaches provide a fundamental approach and solution for metagenomics. The workflow highlighted in green represents the traditional culturing method at the researcher’s discretion. It may allow culturing of some of the possible microorganisms prior to mNGS and metabarcoding. However, this gives an assignment far below sufficient for mNGS and metabarcoding. The stages represented in the figure can be summarized as follows: (a) sampling of infected parts of the plant (leaf discs are preferred); (b) DNA extraction from leaf; (c) library preparation; (d) PCR amplification of gene regions of microbial pathogens with specific gene barcodes; (e) sequencing with Illumina, Nanopore, etc.; (f) bioinformatic analysis of mNGS containing de novo approaches and referenced based assembly, bioinformatic analysis for metabarcoding assembly, clustering, and prediction; and (g) control culture of infected leaves.