Whole-genome single nucleotide polymorphism analysis for typing the pandemic pathogen Fusarium graminearum sensu stricto

Abstract

Recent improvements in microbiology and molecular epidemiology were largely stimulated by whole- genome sequencing (WGS), which provides an unprecedented resolution in discriminating highly related genetic backgrounds. WGS is becoming the method of choice in epidemiology of fungal diseases, but its application is still in a pioneer stage, mainly due to the limited number of available genomes. Fungal pathogens often belong to complexes composed of numerous cryptic species. Detecting cryptic diversity is fundamental to understand the dynamics and the evolutionary relationships underlying disease outbreaks. In this study, we explore the value of whole-genome SNP analyses in identification of the pandemic pathogen Fusarium graminearum sensu stricto (F.g.). This species is responsible for cereal diseases and negatively impacts grain production worldwide. The fungus belongs to the monophyletic fungal complex referred to as F. graminearum species complex including at least sixteen cryptic species, a few among them may be involved in cereal diseases in certain agricultural areas. We analyzed WGS data from a collection of 99 F.g. strains and 33 strains representing all known cryptic species belonging to the FGSC complex. As a first step, we performed a phylogenomic analysis to reveal species-specific clustering. A RAxML maximum likelihood tree grouped all analyzed strains of F.g. into a single clade, supporting the clustering-based identification approach. Although, phylogenetic reconstructions are essential in detecting cryptic species, a phylogenomic tree does not fulfill the criteria for rapid and cost-effective approach for identification of fungi, due to the time-consuming nature of the analysis. As an alternative, analysis of WGS information by mapping sequence data from individual strains against reference genomes may provide useful markers for the rapid identification of fungi. We provide a robust framework for typing F.g. through the web-based PhaME workflow available at EDGE bioinformatics. The method was validated through multiple comparisons of assembly genomes to F.g. reference strain PH-1. We showed that the difference between intra- and interspecies variability was at least two times higher than intraspecific variation facilitating successful typing of F.g. This is the first study which employs WGS data for typing plant pathogenic fusaria.

Two distinct Fusarium graminearum populations colonized European wheat in the past two decades

“…Our study also identified a small A1 population, which consistently with our previous findings [ 20 ], was placed close to the root of the F . graminearum lineage on the phylogenomic tree ( Fig 1B ).…”

“…Among the total of 96 strains used in this study, 82 were chosen to maximize geographic diversity in Europe with regard to the previous phylogenomic approach [ 20 ]. In addition, to enhance the diversity of the sample, we included twelve strains recovered outside of Europe.…”

“…Whole-Genome Sequencing (WGS) was performed as previously described in Kulik et al 2022 [ 20 ]. Briefly, the majority of strains were sequenced by Macrogen, Inc. (Seoul, South Korea) on an Illumina HiSeq X Ten using a paired-end read length of 2 × 150 bp with an insert size of 350 bp.…”

“…graminearum strains, and all known members of the F . graminearum complex [ 20 ]. We found that a phylogenomic clustering of the strains had species specific patterns within this group of cryptic species.…”

A Novel Medium for Isolating Two Japanese Species in the Fusarium graminearum Species Complex and a Dipstick DNA Chromatography Assay for Species Identification and Trichothecene Typing

“…Recently, the whole-genome sequences of all 16 species, including Fusarium sp. CBS 123663, was reported [ 34 , 35 , 36 ], and FGSG_17538 or its homolog was detected in nine species, constituting a subclade in the phylogenomic tree of the Fg complex [ 36 ]. Furthermore, positive amplification by PCR with HS976/HS1039 primers was observed for F. boothii , which belongs to the African clade ( Figure 3 ).…”