Isolation and molecular characterization of clinical and environmental dematiaceous fungi and relatives from Iran

Background and Purpose: The frequency and genetic diversity of black fungi in environmental and clinical settings have not been fully studied in Iran. This study aimed to identify and evaluate intra- and inter-species DNA sequence variation and also understand the phylogenetic relationships of melanized fungi and relatives isolated from different geographical regions of Iran. Materials and Methods: In total, 111 clinical and environmental strains of dematiaceous fungi were isolated, and their internal transcribed spacer ribosomal DNA (rDNA) regions were sequenced and analyzed. Results: An inter-species nucleotide sequence diversity rate of 1 to 464 nucleotides was observed between the species. Intra-species differences were found in the strains of Alternaria alternata, Cladosporium cladosporioides, Alternaria tenuissima, Curvularia spicifera, Aureobasidium pullulans, Curvularia hawaiiensis, Neoscytalidium dimidiatum, Alternaria terricola, Alternaria chlamydospora, Didymella glomerata, and Drechslera dematioidea by 0–59, 0–22, 0–4, 0–4, 0–3, 0–2, 0–2, 0–2, 0–2, 0–1, and 0–1 nucleotide, respectively. Conclusion: The internal transcribed spacer rDNA is useful for the discrimination of several taxa of dematiaceous fungi. However, a better understanding of the taxonomy of species of Alternaria requires a larger rDNA region or a library of other gene sequences.

Introduction ematiaceous fungi are characterized by the presence of pale brown-to-dark melanin-like pigments in the cell wall, which are linked to the pathogenicity of these fungi [1,2]. They comprise a large number of filamentous, yeasts, and yeast-like fungi and relatives, which are found in soil, air, wood, plant, and organic debris [3]. Numerous species in this group are known to cause cutaneous lesions and severe brain encephalitis. Besides, under suitable conditions, they may produce toxins that can pose serious health risks to humans and animals [1,4,5]. Moreover, some of these fungi are of industrial importance and may be used in the production of cellobiose dehydrogenase, citric acid, and pullulan [6].
Despite the increasing importance of dematiaceous fungal infections, little is known about their epidemiology, mode of transmission, or pathogenesis. Epidemiological studies of dematiaceous fungi provide awareness and accurate information on their prevalence. Furthermore, such studies help develop control strategies regarding infections caused by these fungi and improve the diagnosis and development of treatment options [7]. Correct identification to the species level is essential for epidemiological, pathological, toxicological, and industrial purposes, as well as for targeted antifungal therapy [7,8].
For ages, phenotypic methods, including biochemistry, morphology, and physiology have formed the backbone of the identification and taxonomy of dematiaceous fungi [9,10]. Due to the diversities and similarities among different species, morphological features may often be indistinct and inadequate for species identification [10,11]. For accurate identification of these fungi, the focus has shifted towards molecular strategies with the advantages of limited hands-on activity, less required experience, and increased reliability and reproducibility compared with conventional diagnosis [12].
Usage of molecular methods to provide precise and timely information for health professionals is clearly advantageous. Molecular methods, in conjunction with conventional methods or alone, have great potential to develop the analysis of dematiaceous fungi [13]. Nevertheless, various factors, such as nonspecific genetic amplification from other sources (the environment or the host gene), samples containing a mixed infection, an incomplete database particularly related to GenBank, are the limitations to well describe the epidemiology by molecular methods [14].
The DI/D2 domain is not a useful marker for the identification of some species that have identical sequences or an intra-species variation of less than 0.5% [8]. Likewise, sequence data of mtDNA and CHS are not available for all species in GenBank, and partial SSU sequences with little nucleotide variation make the SSU rRNA gene a relatively poor target for discrimination of these fungi [22]. In contrast, phylogenetic analysis and identification of black fungi and relatives based on sequencing of the ITS1 and ITS2 regions has shown to be useful and remains the gold standard target [22].
No study has been performed in Iran about the occurrence and distribution of black fungi in clinical and environmental settings. Therefore, the present research project aimed to identify and evaluate interand intra-species variation within, and also understand phylogenetic relationships of dematiaceous fungi isolated from different geographical parts of Iran. The preliminary data provided in this study could also be useful for improving the differentiation and diagnostic detection of black fungi in the epidemiological, clinical, environmental, and industrial settings.

Samples and fungi isolation
In total, 350 samples, including soil, plant, wood, organic debris, and air were randomly collected from different parts of the center, south, and southwest of Iran, i.e., Shiraz, Bushehr, Isfahan, Ahwaz, and Yasuj cities. This study was approved by the Ethics Committee of Tehran University of Medical Sciences, Tehran, Iran.
Approximately 20 g of each sample (except for air samples) was suspended in 100 mL sterile saline containing 200 U penicillin, 200 μg/ml streptomycin, and 200 μg/ml chloramphenicol. After initial incubation at room temperature for 30 min, 20 mL of sterile mineral oil was added to the solution, followed by vigorous shaking for 5 min. The samples were left for 20 min to let the debris settle down, and the oilwater interphase was carefully collected, inoculated onto the Sabouraud dextrose agar supplemented with 50 mg/L of chloramphenicol (Merck, Germany), Mycobiotic agar (Merck, Germany), home-made potato dextrose agar, and malt extract agar (MEA; Merck, Germany). Afterward, it was incubated for up to four weeks at 28 °C in darkness. The colonies of dematiaceous fungi were then isolated and stored on MEA prior to use [24].
In addition, samples were obtained from bathrooms and washing machines by using sterile cotton swabs moistened with physiological saline which were transported in tubes and inoculated onto MEA agar [25]. Air sampling was performed by the settled plate method using Sabouraud dextrose agar containing chloramphenicol (100 mg/L), gentamicin (40 mg/L), homemade potato dextrose agar, and MEA [26]. Plates were located for 30 min at different heights on the ground. All plates were incubated at 28 °C for at least 4 weeks until the appearance of slow-growing dark colonies [27].
A variety of clinical specimens, including nail, mouth, and sinus samples were collected from patients suspected of fungal infections and submitted to four medical mycology laboratories in Tehran, Isfahan, and Ahwaz, Iran. The fungi were grown on MEA at 28 ºC followed by at least a five-day slide culture and preparation of mount in lactophenol aniline blue. The colonies were studied by observation of the macroscopic morphological features (i.e., growth rate, color, shape, size, and topography) and microscopic examination of the characteristics of the hyphae, conidiophores, conidia, and other conidiation properties [9].

Molecular characterization
Genomic DNA was extracted from the isolated colonies using the glass-bead phenol-chloroform method as previously described [3]. The ITS rDNA  [3,28].

Sequencing and phylogenetic analysis
The PCR products were sequenced in one direction by the primer V9G using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) on an automated DNA sequencer (ABI PrismTM3730 Genetic Analyzer, Applied Biosystems) according to the instructions of the manufacturer. The obtained sequence data were imported into MEGA software (version 6), ambiguous regions were edited manually to improve alignment accuracy, and final identification of isolates was performed by comparing the obtained sequences with the reference sequences of the National Center for Biotechnology Information database.
Sequences were subjected to BioEdit software (version 7.0.5) for pairwise comparisons and multiple alignments to determine intra-and inter-species similarities and differences in nucleotides. The Maximum Likelihood method was applied to the phylogenetic analysis using unambiguously aligned sequences with the Tamura-Nei parameter with substitution model as implemented in the MEGA software (version 7) [29]. Bootstrap values equal to or greater than 70% were considered significant.

Results
Clinical and environmental strains were collected during a 2-year period. In total, 111 strains of potential melanized fungi were isolated out of 350 samples collected from soil, air, and other different environmental sources. Colony characteristics of each colony were studied and subjected to species identification based on ITS-rDNA sequencing.
The mean age and age range of patients were 51 and 27-65 years, respectively. Most patients were female (56%) and in the majority of cases, the infection had been present for a long time. In mycological tests, the characteristic mycelium was seen in direct examination of all nine specimens ( Table 1). The patients resided in Tehran (66.67%), Isfahan (22.22%), and Ahwaz (11.11%), and none of them suffered from any other predisposing diseases ( Table 1).
The environmental strains (n=102) were shown in Table 2. Nucleotide sequences of all isolates were deposited in GenBank under the accession numbers: KY788018-KY788126 and MF422634-MF422636.
Remarkably, species of the order Pleosporales clustered into two clades: Clades 1 and 2. Clade 1 consisted of strains of Alternaria, Curvularia, and Drechslera species while Clade 2 included Didymella as a separate species. The phylogenetic tree revealed Curvularia and Drechslera in Clade 1, forming a subclade closely related to Alternaria species. Phylogenetic analysis showed that Cladosporium species belonged to the branch Capnodiales, forming a single group closely related to Dothidiales order ( Figure. 1).

Discussion
With increasing recognition of the crucial role of fungi in animal and human infections, diagnostic laboratories are expected to be able to quickly detect and accurately identify fungal pathogens to ensure timely and appropriate therapy for infected patients [30]. Lack of pigment or poor sporulation, inter-specific similarities, intra-specific diversity, and variation in growth requirements are some of the features that may influence the precise identification of species. Hence, molecular methods are necessary to distinguish and/or re-classify similar and complex taxa of dematiaceous fungi and discover novel and undescribed species [9][10][11]. Many authors have demonstrated the usefulness of ITS rDNA for species delineation in dematiaceous fungi as the region usually enables discrimination between closely related species [3,21,22]. Therefore, in the present study, ITS sequences were utilized for identification as well as phylogenetic analysis of the isolated dematiaceous fungi.
Based on the findings, Alternaria was the predominant genus in both environmental and clinical samples. The results of the present study are compatible with those of previous reports [31], [32]. However, in a study performed by Parham et al., Ulocladium species were the predominant fungi among all of the dematiaceous fungi, and this finding is not in line with that of the present research [33]. The differences may be due to the source of samples, methods, and other reasons [3].
In the present study, Alternaria species, C. cladosporioides, and C. sphaerospermum were the most commonly observed strains. Differentiation of some species of black fungi, such as Cladosporium species and A. malorum, which are common in both clinical and environmental settings, remains difficult. In this study, isolates identified as Cladosporium species according to the morphological characteristics were recognized as A. malorum based on DNA sequencing. Sequence difference count matrix based on nucleotide pairwise comparison of the ITS region provided evidence showing that this locus was more useful than morphological features for discrimination of these two species.
In a study conducted by Abliz et al., who used the D1/D2 domain for the identification of black fungi, some species of the genus Cladosporium were found to have identical or highly similar sequences with substitutions only at one or two positions [8]. For such species, ITS-rDNA with greater nucleotide variation has a higher potential for discriminating between species than the D1/D2 domain [34,35]. In recent years, DNA-based studies have shown multiple nonmonophyletic genera within the Alternaria complex that do not always associate with species groups based on morphological characteristics.
In the present study, phylogenetic relationships constructed based on sequences of the ITS region from Alternaria isolates and other Pleosporaceae (Ulocladium species, Embellisia species) show the formation of a distinctive clade consisting of A. alternata, A. tenuissima (Alternaria section), A. malorum (Chalastospora section), Alternaria japonica (Japonica section), A. chlamydospora (Phragmosporae section), A. terricola (Ulocladioides section) and Embellisia astragali (Embellisioides section) supported by a bootstrap value of 91%. Results of the present study supported previous observations of the polyphyletic and paraphyletic relationship between Alternaria and the related taxa of Ulocladium and Embellisia.
Among our samples, C. hawaiiensis, C. spicifera, and D. dematioidea were also isolated. Although the genus Curvularia can easily be distinguished from Bipolaris and Drechslera species by sequence analysis, there has been some difficulty in distinguishing them due to their conidial shape, size, and septation. The C. hawaiiensis, C. spicifera, and D. dematioidea went together in our analysis, supported by a bootstrap value of 99%. Moreover, the closely related species, D. glomerata and Ascochyta rabiei formed a separate clade with bootstrap values of 100 ( Figure 1).
In this study, Exophiala phaeomuriformis (belonging to the order Chaetothyriales) and two Ochroconis species were isolated. Ochroconis species Curr Med Mycol, 2021, 7(3): 1-8 7 cause diseases in vertebrate animals and occasionally humans [36]. Phylogenetic analysis based on sequences of the ITS region from Ochroconis isolates indicated that they stand on a separate branch ( Figure  1). The N. dimidiatum and N. novaehollandiae were also among the isolates in the present study. The N. dimidiatum is phylogenetically closely related to N. novaehollandiae [37]. Results of the present research are consistent with those of a study performed by Polizzi et al. [38] which indicated that both species fell into the same clade supported by a bootstrap value of 100%. Sequence variation between strains of dematiaceous fungi led to the observation of clusters with different sections of species. While intra-species sequence diversity of dematiaceous fungi, including A. malorum, Alternaria species, C. sphaerospermum, D. glomerata, and D. dematioidea was low, interspecies nucleotide diversity between most species was quite high. These data advocated that the ITS domain is appropriately variable to be applicable to the identification of several taxa of dematiaceous fungi. The phylogenetic trees constructed from the sequence data revealed that species in the same order segregated into the same cluster.
It was recognized that ITS rDNA sequences do not always provide ample information to differentiate species in the genus Alternaria. The collected data showed a small degree of polymorphism between clinical and environmental isolates as well as a quite low degree of polymorphism within isolates of the same group (non-clinical or clinical group). This might indicate that environmental strains can be a source of human infection. Therefore, more studies on clinical isolates are critical to investigate this issue in greater detail.

Conclusion
In conclusion, identification of black fungi on the basis of morphological characteristics alone is unreliable for the correct determination of species. The ITS sequences were evaluated to be applicable for the identification of several taxa of black fungi. However, for Alternaria species, larger rDNA regions or other gene targets are critical for a better understanding of the taxonomy of this diverse group of fungi.