Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens, leading to yield loss of crops, mycotoxin contamination of food and feed products as well as damage to human and livestock. Human infections of Fusarium spp. are difficult to treat due to broad antifungal resistance by members of this genus. Their role as disease-causing agents in crops and humans suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost effective identification method. Fusarium strains were isolated from food and clinical samples. High-resolution melting curve (HRM) analysis was performed using specific primers targeting internal transcribed spacer (ITS) region, followed with evaluation of specificity and sensitivity. The antifungal susceptibility of four Fusarium species was studied using the Sensititre YeastOne method. HRM analysis revealed reproducible, unimodal melting profiles specific to each of the four Fusarium strains, while no amplification of the negative controls. The minimum detection limits were 100–120 copies based on a 2 µl volume of template. Clear susceptibility differences were observed against antifungal agents by different Fusarium isolates, with amphotericin B and voriconazole displayed strongest antifungal effects to all the tested strains. We developed a simple, rapid, and low-cost qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani). The antifungal susceptibility profiles supplied antifungal information of foodborne and clinical Fusarium spp. and provided guidance for clinical treatment of human infections.


Introduction
As widespread plant pathogens or conditional pathogens of humans, Fusarium spp. can be isolated from soil and plants worldwide [1][2][3]. Fusarium spp. typically infect plant vasculature, disrupting water and nutrient transport to tissues, while also producing toxic secondary metabolites that add to their virulence, cumulatively leading to wilting, rot of various plant organs, and eventually death. In particular, Fusarium oxysporum causes foliar blight, a vascular disease that can kill plants and result in major production losses [4][5][6]. Fusarium solani is a causative pathogen of root rot disease in a variety of crops, and fruit rot in a subset of hosts [7]. Fusarium fujikuroi is a globally distributed pathogen that causes seedborne diseases of rice, such as bakanae disease, through fungal gibberellin production that results in aberrant stem elongation of the host [8]. By contrast, F. lateritium is pathogen of woody plants and trees, and has been isolated from several hosts as a causative agent of wilt, tip or branch dieback, and cankers [9].
In addition, approximately 70 Fusarium spp. are related to infections that cause fusariosis in humans [10,11]. The rate of human fungal infections increases with the upward trend of immunocompromised population, leading to an increase in the morbidity and mortality rates due to fusariosis [12][13][14][15][16]. This could due to the fact that most available antifungal drugs are ineffective against fusariosis [17]. Studies have revealed that flucytosine and echinocandins show poor inhibitory effects against Fusarium species [15,18,19]. Therefore, the antifungal susceptibility characteristics of this genus prone to cause human infections is crucial for the clinical treatment.
Besides its pathogenic effects on plants and humans, Fusarium spp. also produce mycotoxins such as zearalenone, trichothecenes, fumonisin, and fusaric acid, which are dangerous to human and livestock health [7,20]. Symptoms of poisoning can be detected 30-60 min after the ingestion of contaminated corn or wheat. Fusarium mycotoxins do not readily break down through digestion and can be difficult to detect, leading to diagnosis problems. Moreover, detoxification is a clinically complicated process, which makes the monitoring and early identification of fungal contaminants in food a priority for food safety and regulatory agencies [2].
The traditional classification and identification method of Fusarium spp. mainly relies on its morphological characteristics, which is time-consuming and often takes 2-3 weeks to get final result. Molecular identification methods, such as sequencing, multiplex PCR, restriction fragment length polymorphism (RFLP) analysis, multiplex ligation-dependent probe amplification (MLPA), and pyrosequencing, were also used for species identification [17]. Although the above methods are widely used, it takes a long time and may delay the treatment, make the patient's condition worse, or fail to identify the pathogen in time and affect the timeliness of food safety monitoring. Besides, these methods cannot be performed at scale due to the particular requirements for clinicians.
Therefore, a fast, simple, and economical method for practical applications is desirable. High-resolution melting curve (HRM) analysis can be performed in a closed tube right after real-time quantitative PCR (qPCR). Different melting profiles results from various GC contents, DNA sequences, and lengths, facilitating the identification of small differences in the target sequences [21,22]. HRM analysis has been used to successfully identify species from the following: Trichinella (8 species) [23], Candida spp. (23 species) [24], and Aspergillus spp. section Nigri isolates (5 species) [25].
In this study, we reported a qPCR-HRM method that uses a pair of primers to produce different melting profiles, allowing us to identify four important foodborne and clinical Fusarium spp. pathogens (F. fujikuroi, F. lateritium, F. oxysporum, and F. solani) based on differences in the melt profile of the ITS region of rDNA. Antifungal susceptibility of these Fusarium species were studied against 9 commonly used antifungal drugs.

Strain Isolation and Maintenance
Food samples were collected from different cities in China between 2018 and 2021. Foodborne fungi were obtained with a potato dextrose agar (PDA) medium (Land bridge, Beijing, China) and 0.05% chloramphenicol (Sangon Biotech, Shanghai, China) in laboratory conditions. The cultures were isolated into single colony after cultivation at 28°C for 3-4 days, followed by identification of ITS sequencing The primer sequences were as follows (targeting both ITS1, 5.8S and ITS2 regions in the rDNA gene): Alignment of the ITS sequence was performed using National Center for Biotechnology Information (NCBI) nucleotide basic local alignment search tool (BLAST). The samples were preserved and maintained at the National Institute for Communicable Disease Control and Prevention (ICDC, Beijing, China) using 25% glycerol (v/v) at -80°C. The clinical F. solani strain was previously isolated and identified by our lab. The collection sites of strains were recorded as the locations where the food samples or clinical materials were collected.

DNA Extraction
Fusarium spp. samples were all grown for 7 days on Sabouraud's agar (SDA) medium. The cultured cells were transferred into centrifuge tubes with zirconium dioxide beads (U1 mm and U3 mm) and lysis solution (20 mM Tris, 5 mM EDTA, 400 mM NaCl), after which the cells were shaken at low temperature with a biological sample preparation system (Life Real, Hangzhou, China). After cell disruption, tubes were incubated at 95°C for 15 min, followed by cooling at 4°C for 2 h. Supernatant containing crude DNA extraction was transferred into new 1.5 ml tubes after centrifugation at 4°C (12,000 rpm, 10 min), which was subsequently used as amplification templates.

HRM Primers and Plasmids
The ITS region of fungal rDNA bear little pressure of natural selection in the process of evolution, showing a wide range of sequence polymorphisms, which makes ITS sequence suitable for the molecular identification of fungal species [26,27]. Therefore, we assessed the ITS1-ITS2 region sequences from the four Fusarium spp. published in GenBank and designed several primer pairs using the Lasergene Seqman software. Gradient PCR was first performed using the DNA templates previously extracted and agarose gel electrophoresis was used to screen two primers capable of amplifying all four species. The primers sequences are as follows: The alignment of the amplified fragments and primer positions is shown in Fig. 1. Plasmids were synthesized by submitting the sequences of the amplified fragments to Sangon Biotech (Shanghai, China) Co., Ltd.
Establishment of the HRM Method qPCR was carried out in a 30 lL reaction mixture with 11.2 ll ddH 2 O, 0.9 ll of each forward primer and reverse primer, 15 ll HRM analysis mix (TIANGEN, Beijing), and 2 ll DNA template. Conditions for PCR are shown as following: 95°C for 10 min, followed by 35 cycles of 95°C for 15 s, 58°C for 25 s, and 72°C for 25 s. Cooling and heating speeds were 1.6°C/s, while HRM ramping was conducted according to the following: 95°C 10 s, 60°C 1 min, 95°C 10 s, 60°C 1 min, with cooling and heating speeds of 1.6°C/s. Fluorescence data were obtained at 0.025°C increments once a second to produce the melting curves.
Specificity and Sensitivity of HRM Method DNA of commonly found non-Fusarium fungi (including Alternaria spp., Aspergillus spp., Candida spp., and Penicillium spp.) and Fusarium strains were used to perform the qPCR-HRM reaction and identify the specificity of the method.

Antifungal Susceptibility Testing
Antifungal susceptibility testing was carried out using Sensititre YeastOne YO10 panel (ThermoFisher, USA) according to the manufacturer's instructions. In brief, Fusarium isolates were inoculated on PDA medium for 7 days for conidia formation. The conidia were collected with cotton swabs and suspended in 0.85% saline containing 1% polysorbate. Tubes were placed still on a flat surface for 3-5 min to allow heavy particles to sink to the bottom. Conidia suspended in the upper layer was transferred to new tubes followed by vortexing. Spectrophotometry was used to assess conidia suspension by assessing optical density at 530 nm, varying from 0.15 to 0.17. 100 ll inoculum was added to 11 ml RPMI-1640, which was used as final inoculum. 100 ll final inoculum was added to each well of the YO10 plates. These plates were then covered with an adhesive sealing film and incubated at 28°C for 48 h. Finally, the plates were read visually using a reading mirror at the end of 48 h.

Results
The Specificity of the HRM Method for Four Fusarium spp. Complex HRM analysis of ITS amplicons from four Fusarium strains (Table 1) and negative controls revealed melting profiles specific to each of the four strains, while none of the negative controls had amplification   Fig. 2A). By taking the first derivative of the fluorescence signal, we obtained four reproducible, unimodal melting peaks, with different corresponding melting temperatures (Tm) (Fig. 2B). We then used the Tm range from three independent experiments to calculate mean Tm ± SD as a distinct identifier or profile for each isolate to account for inter-assay variability while maximizing sensitivity and reproducibility. The mean Tms for F. oxysporum, F. lateritium, F. fujikuroi, and F. solani were 83.559 ± 0.094°C, 86.145 ± 0.038°C, 86.757 ± 0.044°C and 88.966 ± 0.024°C, respectively. The maximum variation in Tm of the four reference strains was 0.094°C in triplicate assays. Since the mean Tm values for the four strains differed by at least 0.612°C, we concluded that this method could successfully discriminate among the four Fusarium strains.
The Sensitivity of the HRM Method for the Four Fusarium spp. Complex We next conducted HRM analysis using serial dilutions of the ITS1-ITS2 region amplicons from each respective isolate to determine the sensitivity of this method. The results indicated that amplification curves with a characteristic ''S'' shape were generated within a copy number range of * 5 9 10 2 -5 9 10 7 copies/ll (Fig. 3). Standard curves were generated using the Ct values plotted against template concentration (Fig. 4), which indicated that the maximum Ct value in the linear range corresponded to a concentration of 5.24 * 6.33 9 10 2 copies/ll (6.33 9 10 2 copies/ll for F. oxysporum, 5.24 9 10 2 copies/ll for F. lateritium, 6.29 9 10 2 copies/ll for F. fujikuroi, and 5.27 9 10 2 copies/ll for F. solani). These results suggested that the lower limit of detection for this method was 100-120 copies based on a 2 ll volume of template.
The Antifungal Susceptibility of the Four Fusarium spp. Complex To establish antifungal susceptibility profiles for these Fusarium spp. complex members, we tested the resistance of 26 isolates to a panel of antifungal compounds using Sensititre YeastOne YO10 plates. These assays established MIC values for each antifungal agent against the respective isolates, since no breakpoints in antifungal resistance have yet been reported among Fusarium species (Table 2). We observed that F. solani exhibited the strongest drug

Discussion
Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and occasionally reported as opportunistic human pathogens [13,14]. Species within Fusarium are causative agents of disease in a broad range of wild and cultivated plants. Fusaria are also known to produce mycotoxin food contaminants, such as trichothecenes and fumonisins, potentially resulting in severe economic losses for food producers [7]. By contrast, Fusarium spp. are occasionally reported to infect animal hosts as an opportunistic pathogen, resulting in fusarioses, which may be difficult to treat due to broad antifungal resistance by members of this genus [1,12]. In addition, fusarioses is most commonly associated with corneal infections in humans [16]. Thus, their role as disease-causing agents in crops, and to a lesser extent in humans, suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost-effective identification method.
Historically, distinguishing among Fusarium species has proven challenging based only on morphological characterization of isolates, biochemical tests, and disease symptoms [18]. Conventional mycological practices for isolate identification, including morphological characterizations, biochemical assays, and genotyping by high throughput sequencing, multiplex PCR, or RFLP analysis, are time-consuming and laborintensive, and are therefore unsuitable at large scale in clinical laboratories [17]. The HRM method established here relies on monitoring the decrease in dsDNA-binding fluorescent dye signal to find differences in denaturation rate of specific amplified DNA fragments. These denaturation rates indicate binding affinities that are affected by genetic variation at the single nucleotide level [21][22][23][24][25]. Unlike other PCRbased techniques, HRM does not require post-processing steps, such as gel electrophoresis or restriction digests, thus saving time and labor in genotyping. Furthermore, this method can be used to quantify DNA copy numbers in the host sample, and even at very limited initial template concentrations, suggesting its potential for clinical application.
Successful isolate discrimination by HRM depends on design of primers with sufficient specificity and length that the amplicons generate discernibly different melting profiles. Based on these principles, we designed primers for the ITS1-ITS2 region that should amplify 202 bp fragments. Our results showed that the first derivative peaks of melting curves for the four Fusarium spp. strains were both distinguishable by average melting temperature and unimodal. Sequence alignments of the Fusarium spp. complex (Fig. 1) revealed numerous differences in the nucleotide sequences of the amplified fragments among strains, which resulted in the observed distinctions in Tm values and melting curves.
Fusariosis is increasingly recognized as a cause of morbidity or mortality worldwide [16]. In light of the continual growth of immunocompromised populations, this disease is likely to increase in prevalence due to increasing proportion of susceptible, high-risk patients [28]. Indeed, the incidence of Fusarium infections has increased in humans, as have incidence rates of other fungal infections. While the prevalence of Fusarium infections of the pulmonary system is lower than that attributable to Aspergillus, its patient outcomes are generally poorer, with higher rates of morbidity and mortality [15,16]. This is considered to be resulted from the broad resistance of Fusarium species against commonly used antifungal agents [13,20,29]. Thus, in vitro evaluation of antifungal susceptibility of these strains provides significant guidance for appropriate antifungal drug selection in clinic.
Here, we conducted MIC testing for a panel of antifungals, such as amphotericin B, voriconazole, itraconazole, fluconazole, anidulafungin, and caspofungin, among which amphotericin B and voriconazole performed excellent antifungal effects with a mean MIC of 0.89 and 3 lg/ml for F. oxysporum, 1.5 and 4.61 lg/ml for F. solani, 0.12 and 1.67 lg/ml for F. lateritium, 2.67 and 3.33 lg/ml for F. fujikuroi, respectively. A survey examining invasive fusariosis conducted by the European Confederation of Medical Mycology found mean MIC values of 1.51, 7.60, 3.01, and 4.16 mg/L for amphotericin B, posaconazole, voriconazole, and itraconazole, respectively, against a set of 54 clinical isolates [30]. These findings are consistent with our results, which are in agreement with other studies showing that amphotericin B has lower MIC values in vitro than other antifungal compounds [20,29].
In light of the fact that in vitro data do not necessarily correlate with in vivo effects, we emphasize that these MIC results still require validation with clinical data. Notably, despite its high in vitro MIC values, reports suggest that voriconazole is effective in clinical interventions for fusariosis [30]. For instance, Nucci et al. described improved outcomes associated with the use of voriconazole among patients with invasive fusariosis [31]. However, a larger cohort study conducted over a longer observation period, with an expanded panel of isolates is necessary to improve our epidemiological understanding of this infection with robust statistical support. In addition, studies in stem cell lines and solid organ transplantation models are necessary to investigate the local epidemiology of invasive fusariosis.

Conclusion
In this study, we developed a qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani) that provides a relatively simple, fast and low-cost strategy for screening Fusarium isolates. Our results show clear differences in melting curves among these Fusarium species, with high specificity for amplification of the Fusarium spp. ITS1-ITS2 region. In addition, we performed antifungal susceptibility assays, thus providing guidance for the prevention and treatment of human infection with opportunistic Fusarium pathogens.
Author Contributions Resources, data curation and the first draft of the manuscript were performed by Xuexin Hou; methodology, data collection and analysis were performed by Yuanyuan Geng, Rongchen Dai, Fei Zhao and Lihua He contributed to the software, investigation and project administration. Conceptualization, supervision and funding acquisition were performed by Jie Gong. All authors read and approved the final manuscript.

Declarations
Conflict of Interest The authors have no relevant financial or non-financial interests to disclose.

Ethics Approval
No human or animal subjects were required in this study.

Consent to Participate No informed consent was involved in this study.
Consent for Publication All the authors agree to submit the manuscript for possible publication in ''Mycopathologia''.