Molecular identification of Fusarium spp. isolated from wheat based on sequencing of internal transcribed spacer (ITS) region

Molecular identification via Internal Transcribed Spacer region (nrDNA-ITS) sequencing of Fusarium spp. isolates from wheat originated from Stara Zagora region, were performed for the first time in Bulgaria. А total of 60 wheat samples (Triticum aestivum) were morphologically identified at the genus level as Fusarium spp. in advance. The rDNA-ITS region of all isolates was successfully amplified and the PCR products obtained were directly sequenced. After a comparison of detected sequences with NCBI database, members of three different fungal genera (Fusarium, Chaetomium, and Alternaria) were identified. Among Fusarium isolates, the F. tricinctum was prevailing, followed by F. poae. A total of three isolate F. proliferatum, F. graminearum and F. equiseti were presented with a single probe. The lowest genetic distance (0.004) was detected between F. tricinctum isolates. On the base of genetic distances, fungal isolates were grouped in two main clusters – one comprising F. tricinctum isolates and F. proliferatum, and second including F. equiseti, F. graminearum and F. poae. It could be concluded that the rDNA-ITS genome region of the genus Fusarium may be used as a suitable marker of early detection, accurate and reliable identification of Fusarium spp. contamination of wheat. The timely and accurate information would assist in the selection of appropriate approaches for control of fusarium infections and possible mycotoxins contamination of agricultural production.


Introduction
Microscopic fungi from the genus Fusarium are an integral element of soil microflora but are also encountered in underground and aerial parts of plants, plant debris and other organic substrates [1,2]. They induce numerous economically important diseases of crops, e.g. head blight in cereals, Fusarium root rot, corn cob fusariosis, etc., substantially reducing the cereal crop yields and the quality of produce [3][4][5][6]. The combined effect of several Fusarium species, infecting more than one crop at the same time and producing one or more mycotoxins, is an interesting example of biodiversity. During the vegetation and storage, cereals could be attacked by multiple fungal species forming the epiphytic microflora. Fusarium species, causing infections of cereal crops (corn, wheat, barley, oat, etc.) have a dominant position [5,7]. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 21 October 2020 doi:10.20944/preprints202010.0426.v1 A plant may become infected with Fusarium spp. at any stage of its development, depending on both the plant species and the infecting species. Therefore, having in mind that most Fusarium species possess specific mycotoxigenic profiles, the early and accurate identification of Fusarium spp. is essential for predicting the potential risk of infection and prevention of mycotoxin occurrence along the food chain. The unambiguous identification of mycotoxigenic Fusarium species is still an exceptionally critical problem because the number of species in the genus is constantly changing according to various taxonomic systems. A considerable number of Fusarium spp. classifications demonstrated a lack of uniform criteria for identifying species affiliation. Thus, various names are used for the same species or different species are grouped under the same name. The issue is further complicated by the fact that Fusarium species are exceptionally variable and often mutate. Also, many toxigenic strains degenerate and lose toxin-producing ability when stored on artificial nutrient media in laboratory conditions. The information from species recognition criteria used so farmorphological, biological, and phylogenetic species recognition specific for a given region rarely matches [8][9][10]. The main approach to classification of Fusarium spp., which is still independently used, is the morphological one although, in many instances, information provided only by it could be misleading. The reason is the occurrence of some processes in fungi as hybridization, cryptic speciation and convergent evolution [11][12][13]. The independent application of the morphological recognition of microfungi is often limited with regard to a number of morphological features. For example, cultures of some endosymbiotic fungi that routinely produce secondary metabolites, do not exhibit spores. In other spore-forming species, a considerable variation in the shape and size of some asexual structures (conidia, spores) exists within the same strain [14]. In these cases, the exact identification of species affiliation of the respective strain is very challenging. To avoid such limitations, it is advised to combine the morphological approach for identification of microscopic fungi with other contemporary methods of analysis allowed to obtain accurate and consistent results. To this end, a number of mycologists recommend using methods based on DNA sequencing for identification of fungi with high genetic diversity level [15][16][17].
The application of molecular techniques for species recognition of mould fungi dates back to more than two decades with nuclear ribosomal operon primers described by White et al. (1990) [18] including three nuclear ribosomal genes encoding a large ribosomal unit (nrLSU-26S or 28S), a small subunit (nrSSU-18S) and an internal transcribed region functioning as a spacer (ITS1, 5.8, ITS2). The identification of molds on the basis of sequencing, also known as DNA barcoding, is based on the utilization of the nrDNA-ITS region. The meeting of the international consortium of mycologists held in 2011 in Amsterdam has proposed 6 DNA regions from the fungal genome and one of them, namely the ITS region, was officially approved for DNA barcoding marker [19]. Additional fungal studies have provided support for the ITS region as a suitable fungal barcode [20][21][22]. Moreover, most metagenomic studies of fungi are using the ITS region in their studies to identify fungi using modern sequencing technology, which can aid in rapid identification of fungi [23,24,25,26,27,28].
This method for molecular genetic identification requires comparing the studied sequence to the respective reference sequence from a database, e.g. GenBank at the National Center for Biotechnology Information, the European Nucleotide Sequence Archive of the European Molecular Biology Laboratory etc. [29][30].
The ribosomal spacer has a number of advantages which make it a convenient target region for molecular genetic identification of fungi, namely: 1) easy amplification with universal primers, complementary to gene sequences encoding ribosomal DNA molecules; 2) the ITS region is represented as multiple copies (over 200) in the genome as a mosaic of permanent (conserved) and diverse nucleotide sequences allowing for easy amplification even from small amounts diluted DNA or degraded samples; 3) numerous studies demonstrated that the ITS region had a high degree of fungal interspecies variability [31][32][33]. A report in the Journal of Natural Products, containing summarised data for the use of different methods for fungal analysis and the distribution of published research in 2010-2016 showed an increasing tendency for utilisation of nrDNA-ITS as a marker of early fungal species recognition (42%), as compared to independently applied morphological (8%) and other methods [10].
The aim of the present study was to perform molecular identification of Fusarium spp. strains, isolated from wheat originated from Stara Zagora region, Bulgaria with regard to their taxonomy and phylogenesis by sequencing of the nrDNA-ITS region.

Experimental sample collection
A total of 60 wheat samples were analyzed in order to establish the degree of contamination and species from genus Fusarium.  [35].

Mycological examination
One hundred whole grains were collected from each sample after being superficially sterilized with 70% ethanol for 5 min, followed by three items of washing with distilled water to eliminate surface microflora. Grains were placed in 4 Petri dishes (d=15 cm) (25 grains in each) on Czapek Dox medium (MERCK®) (Dimitrov 1983). Inoculated plates were incubated at 22-25°С for 7 days, (BSS 11374/86). After the incubation, the number of grown colonies belonging to the genus Fusarium was counted.
The affiliation of colonies to the genus level was done according to Dimitrov (1983) [37] using the identification keys. The genus level identification included culture examination (colony diameters,  [38]. A suspension of spores was prepared in a 10 ml sterile water blank so that it contains 1-10 spores per low-power (10X) microscope. The water agar medium plates were poured with 0.1 ml of suspension and incubated for 18-20 h at 25°С. Obtained single spore cultures were transferred onto a selective medium (SNA) (Nirenberg 1976 [36]) and incubated at 25°С for 7 days.

DNA extraction and PCR amplification
Genomic DNA from Fusarium spp. strains was isolated from ingrown on SNA plates mycelium with diameter 1±2 cm, initially ground with quartz sand to degrade the polysaccharide cellular wall. The optimum annealing temperature of primers (52.5°С) was preliminarily determined by repeated PCR amplification of the same sample within the temperature range 49.3°÷ 60.7°C.
The obtained PCR products were identified by horizontal electrophoresis on 2% agarose gel diluted in 1×TBE buffer, at 80V/60 min. Agarose gels with incorporated products were observed on МiniBis

Direct sequencing of the region rDNA-ITS
A total of 12 samples (PCR products) were sequenced for the studied nrDNA-ITS region in the Bioneer research lab, South Korea. Sequences for each of samples, provided by Bioneer, were used for subsequent software processing. Bioneer Sequencing Service is a fast and highly reliable service performed on the ABI 3730XL DNA Analyzer, which provides high-quality of sequence analysis data (Phred Score (QV): ≥ 20, Guaranteed read lengths: ≥ 700 bp) within 24 hr from the arrival of sample. We also used Bioneer's oligo synthesis supports for ITS custom primer.

Statistical Analysis
Identification of isolates was performed by comparison of each sequence to respective reference one from the NCBI database through Nucleotide Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). DNA Sequence Assembler v4. (2013) was used for conversion of sequences from "txt" to "Fasta" format.
The established sequences of analysed samples were edited, assembled and aligned by the CLUSTALW tool, using the MEGA7 v.0.26. software [39]. The same software was used to determine genetic distances between studied fungal isolates and phylogenetic tree was reconstructed by the Neighbour-joining method [40][41].

Results
• Mycological examination analysis Mycological examinations of 60 wheat samples harvested in 2018 yielded 12 strains belonging to species from the genus Fusarium. The main mycoflora of wheat samples determined by morphological examination of the fungal isolates (colony characters and microscopically observation of fruit bodies, phiallide, absence of chlamydspores etc.) comprised 9 genera microscopic fungi: Fusarium spp., Aspergillus spp., Penicillium spp., Alternaria spp., Mucor spp., Rhizopus spp., Cladosporium spp., Rhizoctonia spp. and Nigrospora spp. The contamination rate of individual samples with Fusarium spp. ranged from 0-4%, and rates for other genera (Alternaria and Mucor) varied within broader ranges. For the rest of genera, contamination rates varied insignificantly. It should be noted that during 2017-2018, a period characterized by low soil and air humidity during the vegetation of wheat, contamination with different members of the genus Fusarium was expectedly low. Out of the 30 studied wheat samples, 12 (40%) were positive for Fusarium spp., whereas the other 18 (60%) not contaminated with fungi from this genus.

Data analysis from PCR amplification of fungal isolates
The rDNA-ITS region of 12 fungal strains isolated from wheat was successfully amplified by gradient PCR and fragments with the expected length were obtained (Figure 1). The amplicon lengths for Fusarium isolates varied from 515 tо 557 bp. The highest amplified product length was that of Alternaria infectoria -578 bp, while that of Chaetomium spp. was 551 bp.  • Data analysis from the identification of fungal isolates The identification of isolates of studied molds was performed by Nucleotide Blast in NCBI, by comparison of studied sequences to reference sequences from the database. The species affiliation of fungal isolates is presented in Table 1. Among Fusarium isolates, the F. tricinctum was the prevailing species (n = 5), followed by F. poae (n = 2). F. proliferatum, F. graminearum and F. equiseti were presented with a single isolate, which agrees with previous information provided by Beev  • Analysis of genetic distances and phylogenesis of fungal isolates Genetic distances among studied fungal isolates were calculated by MEGA7 v. 0.26 software, on the basis of the number of nucleotide substitutions in analyzed sequences. The lowest genetic distance (0.004) was demonstrated between F. tricinctum isolates, followed by that between F. poae (0.007). The F. proliferatum strain which is differentiated as an independent branch of the phylogenetic tree, was more genetically similar to F. tricinctum than to F. poae, while F. equiseti and F. graminearum exhibited higher genetic identity with F. poae. The cited values are depicted on Figure 3. On the basis of genetic distance values, the phylogenetic tree (Figure 3) was reconstructed using the Neighbour-joining method. The length of phylogenetic tree branches is given with a figure corresponding to genetic distances between studied strains. Neighbour-joining -dendrogram was built on the base of obtained sequences of the region rDNA-ITS of 10 Fusarium isolates from wheat samples in accordance with their reference sequences obtained from NCBI database. The dendrogram ( Figure 3) showed that fungal isolates were grouped in two main clusters -one comprising 5 F. tricinctum isolates and F. proliferatum, and another including 3 isolates of F. equiseti, F. graminearum and F. poae strains.  [43] based on universal ITS primers (ITS-1 and ITS-4). The amplified products (ranged from 522 to 565 bp) have been digested with seven restriction enzymes. A dendrogram derived from PCR-RFLP analysis of the rDNA region has divided the Fusarium isolates into three major groups.

Discussion
In conclusion, the authors pointed out that the assessment of molecular variability based on rDNA RFLP clearly indicated that Fusarium species are heterogeneous and most of the forma speciales have close evolutionary relationships. In order to use the ITS molecular data to identify phytopathogenic form fungus Fusarium, several restriction enzymes specific for each special form that among the regions of the ribosomal cistron, the internal transcribed spacer region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter-and intra-specific variation.

Conclusions
The obtained results in the present study could be affirmed that the primary microflora of the examined samples comprised a total of 9 genera microscopic fungi: Fusarium spp., Aspergillus spp., Penicillium spp., Alternaria spp., Mucor spp., Rhizopus spp., Cladosporium spp., Rhizoctonia spp. and Nigrospora spp. Among Fusarium isolates, the F. tricinctum was prevailing (n=5), followed by F. poae (n=2). F. proliferatum, F. graminearum and F. equiseti were presented with a single isolate. The trend for the occurrence of F. equiseti on wheat was confirmed. The lowest genetic distance (0.004) was detected between F. tricinctum isolates. The F. proliferatum strain was separated as an independent branch of the phylogenetic tree, showing higher genetic similarity to F. tricinctum than to F. poae. The sequencing analysis allowed determinating phylogenetic relationships between studied isolates, which formed 2 clearly distinct phylogenetic tree branches. The results indicated clearly that using genomic rDNA-ITS region was suitable for early detection of wheat seedlings' contamination for the prevention of Fusarium infections.
Author Contributions: Georgi Beev collected the fungal isolates and performed mycological examination. Deyana Gencheva performed DNA analysis. The authors wrote the paper together.

Conflicts of Interest:
All data generated or analysed during this study are included in this published article.The authors declare that they have no competing interests.