Primers were designed for the molecular detection of mycotoxins by targeting their biosynthetic pathway genes such as FUM1 and FUM13 involved in biosynthesis of FB1 using SnapGeneViewer and analyzed with the help of PRIMER-BLAST and BLASTN tools to eliminate, if any non-specific targets in the PCR assay.
Sampling, isolation and identification of Fusarium species
A total of 140 stored maize grain samples were collected from the poultry industries of ten states, Maharashtra (MH), Rajasthan (RJ), Madhya Pradesh (MP), Andhra Pradesh (AP), Uttar Pradesh (UP), Bihar (BH), Tamilnadu (TN), Karnataka (KA), Jharkhand (JK), and Uttarakhand (UK), India.The collected maize samples were analyzed for contamination of Fusarium species on modified Spezieller Nahrstoffarmer agar (SNA) medium . The traditional identification of species of Fusarium was analyzed based on standard manuals and protocols .
Molecular identification of Fusarium species
Genomic DNA/RNA isolation
The genomic DNA/RNA was extracted from Fusarium species after they achieved optimum growth at 72 h. About 100 mg of mycelia were ground to powder in liquid nitrogen using mortar and pestle. According to the manufacturer's instructions, the genomic DNA/RNA was extracted using DNeasy/RNeasy Plant Mini Kit (Qiagen, Germany). The concentration and purity of extracted DNA/RNA were determined using a Nanodrop Spectrophotometer (Thermo Scientific, USA) and stored at -20°C. For reverse transcriptase PCR, total RNA was transcribed using a high-capacity reverse transcription kit (Thermo Fisher Scientific, USA) and an oligo-dT primer. The first-strand cDNA product was used in the PCR with the TEF1-α gene . Further, the species specificity was confirmed with PCR assay using species-specific primer set Vert 1, and Vert 2 primer set specific to F. verticillioides (Supplementary Table 1). The PCR was performed in Applied Biosystem veriti (Thermo Fisher Scientific, USA) with a reaction volume of 20 μL. The amplification mixture consisted of 1 μL of each forward and reversed primers (10 µmol L-1), 1 μL template, 10 μLTaq master mix (Jumpstart, Sigma Aldrich), and 8 μL MilliQ water. The PCR program was set as an initial denaturation at 95ºC for 3 minutes, followed by 35 cycles of 95ºC for 30 s, 52ºC for 30 s, and 72ºC for 1 min, with a final extension at 72ºC for 10 minutes. After successful amplification, the PCR product was purified and sequencing was performed as reported previously .
Molecular detection of FB1gene by PCR assay
The molecular detection of FB1 was carried out by targeting biosynthetic pathway genes such as FUM1and FUM13 respective to FB1 production. The PCR was carried out in a 20 µL reaction mixture containing 50 ng template, 10 μLTaq master mix, 8 μLMilliQ water, 1 μL primers (2 µmol L-1) for each gene. The PCR cycling was set as an initial denaturation at 94 °C for 4 min, followed by 35 cycles at 94°C for 30 sec, annealing at 52°C for 30 sec and extension at 72°C for 1 min with a final extension of 72°C for 10 min. The PCR products were loaded onto 10g L-1 agarose gel electrophoresis and visualized under the gel documentation system.
Pathogenicity of Fusarium species
The effect of Fusarium species on the seed germination, growth, and development of maize was determined using Water-agar (WA) method . Maize seeds were surface sterilized with 0.01% HgCl2 and soaked in 1×106 spores/mL for 12 h on an orbital shaker; the seeds were soaked in sterile MilliQ water without spore suspension was used as control. Following incubation, seeds were transferred into plant tissue culture bottles (43 mm) containing water agar (2%) under aseptic conditions and incubated for 15 d at 25 °C in 16 h light and 8 h dark conditions. At the end of the incubation period, root and shoot inhibition was measured. Furthermore, seed germination was evaluated by using 7days grown culture filtrate (CF) of Fusarium species . Further, FB1 extraction of all the strains of Fusarium was carried out using 50 ml CF of each strain and extracted with equal volume of ACN-MeOH–citrate buffer (12.5/12.5/125, v/v/v) under shaking condition at room temperature for 1 h. The obtained extract was evaporated completely using rotary evaporator (Aditya Scientific, India) and eluted into 200 μl MS grade water. The extract was passed through Sep-Pak column (Waters, Ireland), eluted in 50 μl of MS grade water containing 0.1% formic acid and analyzed for the FB1 detection using LC-MS/MS.
Detection of FB1by LC-MS
An acquity UPLC system (Waters, USA) was used to perform reverse-phase chromatographic partition of the FB1. Peptide BEH C18 column was used for separation with dimensions 2.1mm x 150mm, 1.7μm particle size, and 4 μL injection volume. The column temperature was set at 40ºC, and 8˚ C was the temperature of the sample manager. The separation was carried out with formic acid (0.1%) and water (A); and formic acid (0.1%) and acetonitrile (B) as a mobile phase. An initial gradient used was 90% A and 10% B for 3 min, and then solvent B was increased linearly to 90% within 10 min and was kept constant for 2 min. Further, solvent B was decreased linearly to 10% in 3 min. To avoid carryover in the next acquisition, the column was washed between two acquisitions. The flow rate was 0.5 ml min-1. Eluents were acquired in Mass Spectrometer (TSQ Quantum Access Max mass spectrometer-Thermo Scientific) in positive electrospray ionization (ESI) mode. Each sample was acquired in triplicates. In the ESI+ mode, the MS spray voltage was 4.2 kV. The capillary temperature was 300ºC and probe heating temperature 320ºC with the sheath gas at 45 arbitrary units and the aux gas was 12 arbitrary units.
Genetic diversity of F. verticillioides using Inter Simple Sequence Repeat (ISSR)
The high frequency occurring F. verticillioides isolates were further processed to study the genetic diversity using ISSR markers. The primers were selected based on the polymorphic and reproducible banding patterns to characterize all the F. verticillioides isolates; the 20 different ISSR primers containing di or tri-nucleotide repeats were used in the present study (supplementary table 1). The PCR was carried out in a reaction volume of 20 μL. The amplification mixture consisted of 1 μL of forward and reverse primer each (10 µmol L-1), 10 μLTaq master mix (Jumpstart, Sigma Aldrich), 8 μL of MilliQ water, and 40 ng DNA. Sterile MilliQ water was used as a negative control for each experiment. The PCR program was set as an initial denaturation at 95ºC for 5 minutes, followed by 35 cycles of 95ºC for 30 sec, 44ºC for 45 sec, and 72ºC for 2 min with a final extension at 72°C for 7 min. The PCR amplified products were loaded onto 20g L-1 agarose gel electrophoresis in 1x TAE buffer and visualized under a UV trans-illuminator gel documentation system. The ISSR fingerprint data was analyzed based on the presence or absence of a particular allele indicated as 1 and 0, respectively. However, the pairwise distance among the strains was calculated using Jaccard’s coefficient from the binary matrix. The distance matrices were used to cluster the strains by Unweighted Pair Group Method with arithmetic means (UPGMA). Furthermore, to identify the better threshold microsatellite partitions, the Adjusted Rand value was calculated for each UPGMA/Dice dendrograms to compare each possible combination of microsatellite regions.
Annotation of sequenced TEF1-α gene was performed using BlastN with default parameters against the NR database. The blast hit results were filtered based on Maximum Query Coverage with maximum identity and lowest e-value. Preference was given to F. verticillioides TEF1-α in cases where there was an identical hit from other strains. Phylogenetic analysis was performed using ClustalW of the TEF1-α gene sequences from the isolates. Results of Clustal W were used as input to construct various trees to identify non-F. verticillioides gene clusters. The outliers were removed from the analysis, and distance-based trees were constructed. Interactive Tree of Life online tool was used to construct and visualize trees with Newick format as input for analysis. For ISSR analysis, the presence or absence of amplicon observed at a particular locus was scored as 1 and 0 correspondingly, and pairwise distance among the strains was calculated. Amplicon data was grouped into a 500bp window and provided input to unsupervised hierarchical clustering using the UPGMA method. The resultant file was used to identify Clades based on the distance score. Clustering and visualization were done using MegaX software. The tree file was used as input to the ITOL (Interactive Tree Of Life) online tool and the metadata (Geographical and Virulence) to perform circular tree visualization of the clades.