Molecular Characterization of Fusarium species of food security interest

Project leaders

Graziano Pesole, Elia Choueiri

Agreement

LIBANO - CNRS-L- not in force - National Council for Scientific Research of Lebanon

Call

CNR-CNRS-L 2016-2017

Department

Biomedical sciences

Thematic area

Biomedical sciences

Status of the project

New

Research proposal

The fungal genus Fusarium ranks among the first most important cosmopolitan fungi of soil and plant species. Some species are also known to have a clinical importance as they cause a broad spectrum of infectious disease in human (Salah, et al., 2015). Species members of Fusarium range from saprophytic to plant and human pathogens, where some are producers of mycotoxins (i.e. fumonisins, trichothecenes, zearalenone) (Fu, et al., 2015; Gong, et al., 2015). Saprophytic and pathogenic forms often are found together on the same host plant or in the soil. Mycotoxins production occurs in the presence of suitable host (i.e. plant, animal) and favorable environmental conditions. In addition, the abundance of Fusarium species and the corresponding mycotoxins production depend greatly on the presence of saprophytic Fusarium species or other antagonistic fungi such as Trichoderma sp. (Cha, et al., 2015; El Komy, et al., 2015). Several molecular markers have been investigated to correctly identify Fusarium species and their intraspecific molecular differentiation including producers of mycotoxins. This was inferred by the use of the universal eukaryotic DNA barcode, the internal transcribed spacer one (ITS1) (Wang, et al., 2015), and other markers such as translation elongation factor 1-± (tef-1±), ý-Tubulin and Calmodulin (cal) (Egamberdiev, et al., 2014; Imazaki and Kadota, 2015; Leyva-Madrigal, et al., 2015). With the availability of the next generation DNA sequencing technologies (NGS), it is now possible to accurately profile a complete fungal community by analyzing high-throughput DNA sequencing data generated in a single sequencing run. This innovative approach, denoted as "meta-barcoding", joins the resolution power of an effective molecular marker with high throughput sequencing to recover the maximum information of the biodiversity in a given environment. To achieve an effective and accurate assessment of the microbial population living in the samples under investigation, a suitable representative reference library of the considered molecular marker is mandatory. This is usually done by isolating the single organisms in pure cultures and then specifically sequencing by Sanger method the informative molecular marker chosen for their taxonomic binning.
Only few information are available concerning Fusarium species on plant species in Lebanon (Tabet et al., 2014), where the availability of ITS1 and tef-1± or ý-Tubulin sequences for Fusarium species is almost negligible. Here we propose the construction of a local reference library in Lebanon for Fusarium species by sequencing the ITS1 and tef-1± or ý-Tubulin marker using Sanger method. ITS1 library will first serve to identify Fusarium species present in the country on plant species of high economic impact namely, cereals, banana, fruit trees, vegetables (members of cucurbitaceae and solanaceae) and olive, and in the same time their known potential to produce mycotoxins. Samples of plant organs from symptomatic hosts will be collected from five geographical areas and will be submitted to traditional isolation methods on semi-selective medium. Colonies of Fusarium will be purified and DNA will extracted from mycelium (Hoffman and Winston, 1987) to be used in PCR reactions using ITS1 specific primers.
Moreover, this library will be added to ITSoneDB (Santamaria, et al., 2012), which is a freely accessible curated ITS1 reference database. Data retrieval will concern also all publically available ITS1 sequences in primary (GenBank: NCBI, European Molecular Biology Laboratory:EMBL) and specialized databases (i.e. Fusarium-ID). It will be done by an Application Programming Interface (API) provided by each database (e.g. edirect for NCBI). Then, a custom python script will assess data quality, particularly annotations and sequence identity. Molecular characterization will be achieved by comparing the sequenced Fusarium individuals with reference sequences of Italian and other international Fusarium isolates. Similarity search will be performed in order to flag the sequenced individuals onto the closest reference species. Furthermore, the maximum likelihood phylogenetic analysis of the classified sequences will be done to determine the evolutionary relationship among all identified species including their references.
The additional markers, namely tef-1± or ²-tubulin will be considered to study the intra-specific variability of assigned species. This includes SNP (Single Nucleotide Polymorphism) calling analyses, on individuals of the same species or those closely related belonging to the same phylogenetic clade, to summarize the intra-specific genetic variability and the potential use of these markers in future molecular typing studies and mycotoxins' traceability. Marker choice will depend on a preliminary in-silico study aiming at the identification of the best marker correlated with mycotoxins production. In addition, other metadata such as host species, geographical origin, reference strains and fungicides resistance will be integrated with molecular data (inter-and intra-specific groups) to find the associated genetic variants with the discrete variables. For this purpose, sampling in the second year will focus on areas in which Fusarium isolates known as mycotoxin producers or biological control agents were recovered during the first year and identified based on their ITS1 sequence. The produced sequences will be deposited in NCBI, and the analyses will be published on a peer-reviewed international scientific journal.
The present research, if suitably accomplished, will provide the scientific community a more comprehensive molecular data on Fusarium species in Lebanon and in the Middle East. In addition, it will enrich the reference library of ITS1 in ITSoneDB, which is an important issue in meta-barcoding studies and will help us to optimize NGS data analysis methods in our research expertise area.

Research goals

1- Characterization of Lebanese Fusarium isolates molecular types and flagging them for potential mycotoxins production or for their use as biological control agents (in the case of saprophytic species).
2- Construction of a reference ITS1 library and one additional molecular marker (tef 1-± or ²-tubulin) of a representative collection of Lebanese Fusarium isolates coming from different plant species. The produced sequences will be deposited in NCBI to be available for the scientific community.
3- Enrichment of the ITSoneDB reference database with Fusarium species ITS1 sequences with their associated metadata. This includes first the building of Fusarium reference database by retrieving the corresponding molecular markers sequences from primary (NCBI, EMBL) and specialized databases (Fusarium-ID), as well as by adding the entries provided by our collaboration with ISPA-CNR.
4- In-silico testing of tef 1-± or ²-tubulin molecular marker for their potential use in Fusarium intra-specific typing based on ITS1species assignment.
5- Determine the evolutionary relationship between the sequenced isolates and the reference ones through phylogenetic analyses. Metadata mapping on phylogeny to evaluate the association between genetic variability and discrete variables.
6- Publish the study on an international peer-reviewed journal.

Last update: 27/09/2024