The frequency distribution of Aspergillus section Nigri from clinical and environmental samples in Iran

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Although most of these species are known for food spoilage and some have industrial applications, some of these species, including Aspergillus tubingensis , A. welwitschiae , and A. niger , are etiologic agents of human and animal aspergillosis. Moreover, the frequency of each species in different environments is correlated with meteorological conditions. The present study aimed to investigate the frequency of Aspergillus section Nigri in clinical samples and environmental sources in several Iranian provinces. Methods A total of 629 samples, including 261 clinical materials and 368 environmental materials, were collected from seven Iranian provinces. The Aspergillus section Nigri was initially screened on the basis of colony morphology and then subjected to sequencing via the calmodulin ( CaM ) gene. Maximum likelihood phylogenies were constructed via MEGA 11 software. Results Of the 192 Aspergillus section Nigri isolates , 41.15% were identified as A. tubingensis , followed by A. welwitschiae (35.94%), A. niger ( sensu stricto ) (17.71%), A. neoniger (1.56%), A. piperis (1.56%), A. aculeatus (1.04%), and A. luchuensis (1.04%). The most prevalent species were A. tubingensis and A. welwitschiae from clinical and environmental samples, respectively. In addition, the frequency of these species varied across Iranian provinces. Conclusions This study determined the presence and frequency, distribution, and phylogenetic relationships of eight black aspergilli, A. tubingensis , A. welwitschiae , A. niger , A. neoniger , A. piperis , A. aculeatus , and A. luchuensis , associated with clinical and environmental samples from several Iranian provinces. In terms of the source of isolation, A. tubingensis was most common in the soil and air samples, A. welwitschiae was most common in the otomycosis and bronchoalveolar lavage (BAL) samples, and A. tubingensis was most common in the sputum samples. Aspergillus section Nigri A. tubingensis A. welwitschiae Molecular identification Calmodulin gene Iran Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aspergillus section Nigri (black aspergilli, due to the production of black spores) is an important fungal species in biotechnology research and medical and food mycology ( 1 – 3 ). They are distributed worldwide, with the main source being soil. Moreover, this species has also been isolated from many fruits, vegetables, various grains, beans, nuts, dairy products, and meat products ( 4 ). The taxonomy of Aspergillus section Nigri has been revised in several periods on the basis of molecular phylogenetic methods and has continuously evolved. The last taxonomy revision included more than twenty different species ( 5 ). Aspergillus tubingensis , A. niger ( sensu stricto ), A. welwitschiae , A. awamori , A. aculeatus, A. acidus , A. piperis , A. neoniger , A. luchuensis , A. carbonarius , and A. brasiliensis are some of the well-known species described in this section ( 3 ). Many species in this area have effective products in the food and biotechnology industry, such as vitamins, antibiotics, and hydrolytic enzymes ( 6 ). Aspergillus section Nigri has been regarded as a benign fungus (GRAS, generally regarded as safe) by the FDA, and the clinical implications of species such as A. uvarum , A. awamori , and A. foetidus have rarely been reported ( 4 , 7 ). However, in recent years, the incidence of infections caused by black aspergilli species, including A. niger, A. welwitschiae and A. tubingensis , has increased ( 8 , 9 ). They are known not only as the cause of noninvasive mycoses (otomycosis, keratitis, onychomycosis, allergic aspergillosis, and aspergilloma) but also as invasive aspergillosis ( 7 , 8 , 10 ). Several meteorological conditions, as well as vegetation, affect the distribution of fungi in the environment. As a result, the habitat niches of saprophytic fungi differ across areas and could reflect the etiologic agents of mycosis. Iran's climate ranges from arid or semiarid and subtropical to the Caspian coast and northern forests. Each condition has a specific habitat niche with different frequencies of fungi. The purpose of the present study was to discriminate Aspergillus section Nigri , which was isolated from clinical and environmental samples in some Iranian provinces, via the calmodulin gene. Moreover, in this study, we determined the frequency of this species in several provinces of Iran. Materials and methods Isolates and primary identification Environmental samples, including air and soil samples, were collected from seven provinces in Iran (Khuzestan, Tehran, Alborz, Kermanshah, Kohgiluyeh, Boyer-Ahmad, Fars, and Esfahan). Clinical samples were also collected from bronchoalveolar lavage (BAL), sputum, and otomycosis lesions from three provinces in Iran (Khuzestan, Kohgiluyeh, and Boyer-Ahmad and Fars) (Fig. 1 and Table 1 ). The otomycosis samples were obtained from patients attending the otolaryngology clinics via sterile swabs and directly cultured on suitable culture media. Bronchoalveolar lavage and sputum samples were collected from patients who were referred to the bronchoscopy department affiliated with Ahvaz Jundishapur University of Medical Sciences via standard techniques outlined by Costabel et al. ( 11 ). The samples were preserved in sterile containers without any preservative components and transferred to our laboratory. Sputum and BAL samples were prepared via standard methods and cultured on suitable culture media. Soil samples (0.5 g) were suspended in 10 mL of sterile distilled water containing chloramphenicol (Bio Basic, Canada) and allowed to sediment for 1 h. A total of 0.1 mL of the supernatant from each sample was cultured. All the clinical and environmental samples were cultured on Sabouraud dextrose agar (SDA) supplemented with 0.5 µg/ml chloramphenicol (Condalab, Spain) and incubated at 35°C for 1‒2 weeks. Moreover, SDA plates with chloramphenicol were used for trapping airborne fungal spores. Table 1 Geographical locations of the clinical and environmental samples. Geographical origin Environmental samples Clinical samples Total Soil Air Otomycosis BAL Sputum Khuzestan 115 55 87 103 13 373 Kohgiluyeh and Boyer-Ahmad 40 15 54 - - 109 Alborz 25 40 - - - 65 Fars 30 - - 3 1 34 Kermanshah 23 - - - - 23 Tehran 20 - - - - 20 Esfahan 5 - - - - 5 Total 263 110 141 106 14 629 Colonies with brown to black characteristics were initially screened as Aspergillus section Nigri ( 4 ). Colonies were purified on fresh SDA and then identified on the basis of conventional morphological features, including colony characteristics and the microscopic morphology of each isolate, such as vesicle type, phialide, and conidial arrangement ( 4 , 9 ). Moreover, a fresh and pure culture of each isolate was prepared and stored at ambient temperature for subsequent investigations. DNA extraction and molecular identification Strains were cultured on SDA (BioLife, Italy) and incubated at 30°C for 3 days. The genomic DNA of the strains was extracted via the method described by Makimura et al. ( 12 ), with some modifications. A small amount of mycelia was crushed with a conical grinder in 500 µL of lysis buffer for 30–60 s. Then, 150 µL of lithium acetate (CDH, New Delhi, India) was added, and the mixture was centrifuged twice at 12000 × g for 3 min (at 4°C). The supernatant (DNA) was extracted by adding an equal volume (400–500 µL) of isopropanol (Merck, Germany) at -20°C for 10 min and then centrifuging at 12000 × g for 3 min (4°C). The isopropanol was discarded, and the DNA was washed with 300 µL of 70% ethanol at 12000 × g for 10 min (at 4°C). Finally, the ethanol was discarded, and the DNA was dried and suspended in 50 µL of ultrapure water. The purified DNA was stored at − 20°C until subsequent analysis. The partial sequence of the calmodulin gene (CAM) was amplified with the primers CMD5 (5-CCGAGTACAAGGAGGCCTTC-3) and CMD6 (5-CCGATAGAGGTCATAACGTGG-3) ( 13 ). Sequence analysis and phylogenetic analysis The PCR products were subsequently sequenced with the forward calmodulin primer (CMD5) by the Cardio-Genetic Research Center sequence service (RCMRC, Tehran, Iran). The sequences were subsequently aligned via MEGA software (version 11), and the species were identified via searches of databases via the online BLAST system of the NCBI ( http://www.ncbi.nlm.nih.gov ). All sequences were submitted to DDBJ ( https://www.ddbj.nig.ac.jp ), and accession numbers were obtained. Phylogenetic relationships between Aspergillus section Nigri strains were constructed via the maximum likelihood method with MEGA software (version 11) and analysed to validate clade assignments. Results Among the 629 clinical and environmental samples, 306 (48.65%) showed pure growth of Aspergillus section Nigri species. Moreover, 323 (51.35%) yielded other fungi and some bacteria, which were not included in the study. Among the 261 clinical samples, only 131 isolates were identified, and among the 368 environmental samples, only 175 Aspergillus section Nigri isolates were identified (Table 2 ). Finally, 192 isolates (118 and 74 clinical and environmental isolates, respectively) were selected for sequence analysis. Table 2 Frequencies of positive cultures from clinical and environmental samples. Samples No. Positive Clinical (261) Otomycosis 141 110 (78.01%) BAL 106 19 (17.92%) Sputum 14 2 (14.28%) Environmental (368) Air 110 26 (23.63%) Soil 258 149 (57.75%) Total 629 306 (48.65%) Sequencing of Aspergillus section Nigri Among the 192 isolates of Aspergillus section Nigri , 79 (41.15%) were identified as A. tubingensis , followed by A. welwitschiae (69, 35.94%), A. niger ( sensu stricto ) (34, 17.71%), A. neoniger (3, 1.56%), A. piperis (3, 1.56%), A. aculeatus (2, 1.04%), and A. luchuensis (2, 1.04%). The accession numbers for the environmental and clinical isolates are shown in Table 3 . Table 3 GenBank accession numbers of Aspergillus section nigri strains isolated from environmental and clinical samples. Species Environmental isolates Total Clinical isolates Total A. tubingensis LC794827–64 38 LC813269–82 LC830486, LC830488, LC830492, LC830494 – LC830495, LC830499, LC844950, LC844953, LC844955, LC844957–8, LC844960–1, LC844965, LC844967–8, LC844970, LC844972–4, LC844977, LC844979–80, LC844984, LC844987–8, LC844995 41 A. welwitschiae LC794865–77, LC794808–811 17 LC813244–68, LC830485, LC830487, LC830489–90, LC830493, LC830497, LC830500–1, LC830503, LC844943, LC844947, LC844949, LC844951, LC844954, LC844959, LC844962–64, LC844966, LC844969, LC844971, LC844981–3, LC844985, LC844993, LC844996 52 A. niger LC794812–25 14 LC813238–43, LC844944–46, LC844948, LC844956, LC844975–6, LC844978, LC844986, LC844989–92, LC844994 20 A. neoniger LC794806–7 2 LC844952 1 A. piperis LC794826 1 LC830491, LC830498 2 A. aculeatus LC794804–5 2 – 0 A. luchuensis – 0 LC830496, LC830502 2 Total 74 118 Frequency distribution of isolates in Iranian provinces The details of Aspergillus section Nigri isolated from clinical and environmental samples are shown in Table 4 . Our results indicated that A. welwitschiae was the most common isolate from clinical materials in Khuzestan, followed by A. tubingensis , whereas A. tubingensis was the most common strain, followed by A. welwitschiae in Kohgiluyeh and Boyer-Ahmad (Fig. 2 ). Moreover, the environmental isolates shown in Fig. 3 clearly indicate that A. tubingensis was the most common environmental isolate of Aspergillus section Nigri in all the sampled provinces except Esfahan. Table 4 Details of Aspergillus section Nigri from clinical and environmental samples. Aspergillus Species Environmental isolates Clinical isolates Total Soil Air Total Otomycosis Sputum BAL Total A. tubingensis 26 12 38 36 2 3 41 79 A. welwitschiae 12 5 17 45 0 7 52 69 A. niger 10 4 14 14 0 6 20 34 A. neoniger 1 1 2 1 0 0 1 3 A. piperis 0 1 1 2 0 0 2 3 A. luchuensis 0 0 0 0 0 2 2 2 A. aculeatus 1 1 2 0 0 0 0 2 Total 50 24 74 98 2 18 118 192 Phylogenic analysis The maximum likelihood phylogenetic analysis of Aspergillus section Nigri isolated from clinical and environmental samples is shown in Fig. 4 . 79 A. tubingensis , 3 A. piperis , 3 A. neoniger , 2 A. aculeatus , and 2 A. luchuensis strains representing clade 1. Clade 2 contained 2 subclades, A and B, with clade A containing 51 A. welwitschiae and clade B containing 34 A. niger and 18 A. welwitschiae . Discussion Understanding the regional distribution of Aspergillus section Nigri species and their antifungal susceptibility patterns is crucial for monitoring the emergence of antifungal resistance across various species. For several decades, more studies have shown that A. niger is the predominant etiologic agent of otomycosis, with a worldwide distribution ( 14 – 16 ). However, with respect to the exact differentiation of Aspergillus section Nigri and the new taxonomy, new species were identified and presented. Our study revealed the frequency of clinical and environmental strains of Aspergillus section Nigri in several Iranian provinces. Aspergillus welwitschiae (44.07%) was the most common strain isolated from clinical samples, followed by A. tubingensis (34.75%) and A. niger (16.95%). Similar studies from China ( 9 ) and France ( 17 , 18 ) have shown that A. welwitschiae is the most common isolate from clinical samples, followed by A. tubingensis and A. niger . Moreover, Halvaeezadeh et al. obtained the same results that confirmed our results ( 19 ). Importantly, A. welwitschiae is closely related to A. niger ; thus, it may be misidentified as A. niger via ITS region analysis ( 1 ). Although A. welwitschiae is the most common clinical isolate, A. welwitschiae and A. tubingensis are common clinical isolates from Khuzestan and Kohgiluyeh- Boyer-Ahmad, respectively. In recent years, several reports of chronic pulmonary aspergillosis or acute invasive aspergillosis caused by A. welwitschiae have been published from Asian and Middle Eastern countries ( 20 , 21 ). Recently, three cases of COVID-19-associated pulmonary aspergillosis (CAPA) caused by A. welwitschiae were reported in immunocompetent patients in France ( 22 ) and Brazil ( 23 ), and one case was reported in a Portuguese immunocompromised patient ( 21 ). Najafzadeh et al. identified Aspergillus species ( A. tubingensis , A. welwitschiae , A. fumigatus , A. sydowii , A. neoniger , and A. terreus ) as the main causes of various infections, such as invasive aspergillosis (IA) in immunocompromised patients, and presented these infections with high mortality rates ( 24 ). In another study, A. welwitschiae was found to be the main cause of secondary pneumothorax in patients with a lung transplant. Fukushima et al. also demonstrated that this case highlights the potential of A. welwitschiae to cause sudden pulmonary infection, presenting as pneumothorax, despite prophylactic treatment with itraconazole ( 25 ). In a case study, A tubingensis was identified as the cause of a solitary brain abscess in a Japanese man with alcoholic liver cirrhosis without any immunosuppressive disease ( 26 ). Our study revealed that 38.9% of the ball isolates contained A. welwitschiae . Notably, the presence of A. welwitschiae usually indicates resistance to antifungal azole. According to previous studies, to the best of our knowledge, the initial resistance mechanism is attributed to the overexpression of the CYP51A gene, which is a major challenge for responding to treatment in patients ( 9 , 17 , 25 ). In addition, unfortunately, Aspergillus section Nigri has been reported to have lower susceptibility to voriconazole (VOR), the first choice for treating invasive aspergillosis ( 9 , 20 , 26 ). Like other Aspergillus species, Aspergillus section Nigri is a common saprophytic fungus in the environment. The environment plays an important role in the epidemiology of invasive aspergillosis. Several metrological conditions, as well as vegetation, play important roles in the mycoflora and distribution frequency in the environment. A study by Barberis et al. was conducted to identify the general correlation of the distribution of the Aspergillus group from Argentina on vineyard soils. In this study, A. niger aggregates were the prevalent group ( 27 ). Aspergillus welwitschiae was isolated by Li et al. in China (2023) and identified as the causative agent of postharvest rot on Chinese cabbage ( 28 ). In addition, the presence of three black aspergilli species, A. tubingensis , A. welwitschiae , and A. ovarum , was reported in central Italy ( 29 ). Moreover, A. welwitschiae and A. tubingensis are predominant in the soil, grapes, and air in the Vineyard, respectively ( 30 ). The results of the phylogenetic analysis of the isolates revealed that the calmodulin gene is highly able to identify Aspergillus section Nigri . As shown in Fig. 4 , clade 1 (clade A. tubingensis ) contained A. tubingensis , A. piperis , A. neoniger , A. aculeatus , and A. luchuensis strains. Clade 2 (clade A. niger aggregate) contained the closely related species A. niger and A. welwitschiae , which is similar to the results of Marques et al. ( 30 ). Moreover, Najafzadeh et al. phylogenetically analysed Aspergillus section Nigri and reported A. niger and A. welwitschiae in a clade that was completely separated from clade A. tubingensis ( 24 ). Conclusion Overall, knowledge of Aspergillus section Nigri species distribution plays a crucial role in proper therapy. In summary, this study determined the presence of eight black aspergilli, A. tubingensis , A. welwitschiae , A. niger , A. neoniger , A. piperis , A. aculeatus , and A. luchuensis , associated with clinical and environmental samples in Iran. As mentioned in the results, A. tubingensis , A . welwitschiae , and A. niger were by far the predominant species. In terms of the source of isolation, A. tubingensis was most common in the soil samples as well as in the air samples, A. welwitschiae was most common in the otomycosis samples as well as in the BAL samples, and A. tubingensis was most common in the sputum samples. Abbreviations A: Aspergillus BAL: Bronchoalveolar lavage FDA: Food and Drug Administration SDA: Sabouraud dextrose agar DNA: Deoxyribonucleic acid PCR: Polymerase chain reaction Declarations Ethics approval and consent to participate The ethics committee of Ahvaz Jundishapur University of Medical Sciences approved this project under reference no. IR.AJUMS.MEDICINE.REC.1402.020. The study was carried out in accordance with the Helsinki Rules. Additionally, written informed consent was obtained from all patients and parents (for children) involved in any experimental work with humans. Consent for publication Not applicable Availability of data and materials All the data have been included in the manuscript. However, raw data will be made available upon request from the corresponding author via email ( [email protected] ). Competing Interests The authors declare that they have no competing interests. Funding This study was funded by Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (grant number OG- 0208). Authors' contributions S. H. wrote the original draft, software, methodology, formal analysis, data curation; GA. J., SH. B., and M. K. resources, visiting patients, data curation; M. G.and H. J. Resources, preparing environmental samples; A. Z. M. writing, review & editing, supervision, project administration, data interpretation, funding acquisition, conceptualization. Acknowledgements The authors express their gratitude to Jalal Hamzehee for collecting many of the study samples, as well as the bronchoscopy section and otolaryngology clinic of Imam-Khomeini Hospital, Ahvaz University of Medical Sciences, for the facilities provided to carry out the research project. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6994062","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484982384,"identity":"2ce15247-8ab5-4460-ad36-40612fafda5d","order_by":0,"name":"Sara Hamzehee","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Sara","middleName":"","lastName":"Hamzehee","suffix":""},{"id":484982385,"identity":"b3389c1a-28e2-4c19-926d-1dc23866f3aa","order_by":1,"name":"Maral Gharaghani","email":"","orcid":"","institution":"Yasouj University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maral","middleName":"","lastName":"Gharaghani","suffix":""},{"id":484982386,"identity":"d93f5527-9c81-4d4c-80b5-48ddb09deb59","order_by":2,"name":"Seyed Hamid Borsi","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Hamid","lastName":"Borsi","suffix":""},{"id":484982387,"identity":"745295cd-dfba-4b6f-ba16-263ae57d9f52","order_by":3,"name":"Hadis Jafarian","email":"","orcid":"","institution":"Shiraz University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hadis","middleName":"","lastName":"Jafarian","suffix":""},{"id":484982388,"identity":"59e16887-8349-4471-b0ec-8303bbf05088","order_by":4,"name":"Gholam Ali Jalaee","email":"","orcid":"","institution":"Apadana Hospital","correspondingAuthor":false,"prefix":"","firstName":"Gholam","middleName":"Ali","lastName":"Jalaee","suffix":""},{"id":484982389,"identity":"fad278ad-2624-443c-a2b2-f868642455df","order_by":5,"name":"Maryam Kardooni","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Kardooni","suffix":""},{"id":484982390,"identity":"2044cdd8-f299-42ca-aad3-3d52e0b1bab8","order_by":6,"name":"Ali Zarei-Mahmoudabadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIie3QMQ6CMBSA4RKSsjRhfQOBK9SQGAcOU2JSFkm8ghODGK/SzTg3xgV2CAsuTC5uDMQIrJpaN4f+U4d+ea9FyGT61xgBn84nutIlXhRSZE8EdMdEPBYzQRokyMpb225kcnKOF9FvAbnZnimJlSchZYVMz7nEdT4uBkUplMRGHEOcy1RUa1yRkVBI1QS7ndPHT5nQkdSDDiHA8fjJnE2k0ZoC0NnASLQQhVw2HgXy9S3BkVuPnkBAr7uuvg+R72YHNXnf9LfrJpPJZPrYCy/NQtLSR3XtAAAAAElFTkSuQmCC","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Ali","middleName":"","lastName":"Zarei-Mahmoudabadi","suffix":""}],"badges":[],"createdAt":"2025-06-27 19:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6994062/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6994062/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-025-04323-7","type":"published","date":"2025-09-01T15:57:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86862489,"identity":"bb24ec92-73ac-4a01-83fa-de543c008653","added_by":"auto","created_at":"2025-07-16 12:35:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120149,"visible":true,"origin":"","legend":"\u003cp\u003eGeographical locations of sampling in Iran (AL; Alborz, KE; Kermanshah, KO; Kohgiluyeh and Boyer-Ahmad, KH; Khuzestan, ES; Esfahan, and FA; Fars)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6994062/v1/eb5b4f818c074998d84042fb.png"},{"id":86862490,"identity":"a187233d-af76-46c0-bf0c-697b4217d837","added_by":"auto","created_at":"2025-07-16 12:35:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29151,"visible":true,"origin":"","legend":"\u003cp\u003eThe frequency of clinical \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e from Iran\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6994062/v1/633cb9c811ab1ab624a9af4b.png"},{"id":86862496,"identity":"44783111-be83-4630-b161-9e629827b393","added_by":"auto","created_at":"2025-07-16 12:35:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":255600,"visible":true,"origin":"","legend":"\u003cp\u003eThe frequency of environmental \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e from Iran\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6994062/v1/4afedb5b8cf35ca38ec8c690.jpeg"},{"id":86862495,"identity":"367cbd20-5050-49c6-93f4-d48c9380cbac","added_by":"auto","created_at":"2025-07-16 12:35:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2593763,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood species tree based on calmodulin (\u003cem\u003eCaM\u003c/em\u003e) sequences, showing the relationships between \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6994062/v1/0b2de9a670acac41123bbed4.jpeg"},{"id":90827980,"identity":"62083625-8294-4908-af92-1f16e3af450b","added_by":"auto","created_at":"2025-09-08 16:04:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3897097,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6994062/v1/2456cafe-6235-4f4f-890f-c02d89249863.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The frequency distribution of Aspergillus section Nigri from clinical and environmental samples in Iran","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e (black aspergilli, due to the production of black spores) is an important fungal species in biotechnology research and medical and food mycology (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). They are distributed worldwide, with the main source being soil. Moreover, this species has also been isolated from many fruits, vegetables, various grains, beans, nuts, dairy products, and meat products (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The taxonomy of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e has been revised in several periods on the basis of molecular phylogenetic methods and has continuously evolved. The last taxonomy revision included more than twenty different species (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). \u003cem\u003eAspergillus tubingensis\u003c/em\u003e, \u003cem\u003eA. niger\u003c/em\u003e (\u003cem\u003esensu stricto\u003c/em\u003e), \u003cem\u003eA. welwitschiae\u003c/em\u003e, \u003cem\u003eA. awamori\u003c/em\u003e, \u003cem\u003eA. aculeatus, A. acidus\u003c/em\u003e, \u003cem\u003eA. piperis\u003c/em\u003e, \u003cem\u003eA. neoniger\u003c/em\u003e, \u003cem\u003eA. luchuensis\u003c/em\u003e, \u003cem\u003eA. carbonarius\u003c/em\u003e, and \u003cem\u003eA. brasiliensis\u003c/em\u003e are some of the well-known species described in this section (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMany species in this area have effective products in the food and biotechnology industry, such as vitamins, antibiotics, and hydrolytic enzymes (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e has been regarded as a benign fungus (GRAS, generally regarded as safe) by the FDA, and the clinical implications of species such as \u003cem\u003eA. uvarum\u003c/em\u003e, \u003cem\u003eA. awamori\u003c/em\u003e, and \u003cem\u003eA. foetidus\u003c/em\u003e have rarely been reported (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, in recent years, the incidence of infections caused by black aspergilli species, including \u003cem\u003eA. niger, A. welwitschiae\u003c/em\u003e and \u003cem\u003eA. tubingensis\u003c/em\u003e, has increased (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). They are known not only as the cause of noninvasive mycoses (otomycosis, keratitis, onychomycosis, allergic aspergillosis, and aspergilloma) but also as invasive aspergillosis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral meteorological conditions, as well as vegetation, affect the distribution of fungi in the environment. As a result, the habitat niches of saprophytic fungi differ across areas and could reflect the etiologic agents of mycosis. Iran's climate ranges from arid or semiarid and subtropical to the Caspian coast and northern forests. Each condition has a specific habitat niche with different frequencies of fungi. The purpose of the present study was to discriminate \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e, which was isolated from clinical and environmental samples in some Iranian provinces, via the calmodulin gene. Moreover, in this study, we determined the frequency of this species in several provinces of Iran.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eIsolates and primary identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEnvironmental samples, including air and soil samples, were collected from seven provinces in Iran (Khuzestan, Tehran, Alborz, Kermanshah, Kohgiluyeh, Boyer-Ahmad, Fars, and Esfahan). Clinical samples were also collected from bronchoalveolar lavage (BAL), sputum, and otomycosis lesions from three provinces in Iran (Khuzestan, Kohgiluyeh, and Boyer-Ahmad and Fars) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The otomycosis samples were obtained from patients attending the otolaryngology clinics via sterile swabs and directly cultured on suitable culture media.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBronchoalveolar lavage and sputum samples were collected from patients who were referred to the bronchoscopy department affiliated with Ahvaz Jundishapur University of Medical Sciences via standard techniques outlined by Costabel et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The samples were preserved in sterile containers without any preservative components and transferred to our laboratory. Sputum and BAL samples were prepared via standard methods and cultured on suitable culture media. Soil samples (0.5 g) were suspended in 10 mL of sterile distilled water containing chloramphenicol (Bio Basic, Canada) and allowed to sediment for 1 h. A total of 0.1 mL of the supernatant from each sample was cultured. All the clinical and environmental samples were cultured on Sabouraud dextrose agar (SDA) supplemented with 0.5 \u0026micro;g/ml chloramphenicol (Condalab, Spain) and incubated at 35\u0026deg;C for 1‒2 weeks. Moreover, SDA plates with chloramphenicol were used for trapping airborne fungal spores.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGeographical locations of the clinical and environmental samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGeographical origin\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003eEnvironmental samples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eClinical samples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSoil\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAir\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOtomycosis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBAL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSputum\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKhuzestan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e103\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" 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align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKermanshah\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTehran\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEsfahan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e263\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e629\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eColonies with brown to black characteristics were initially screened as \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Colonies were purified on fresh SDA and then identified on the basis of conventional morphological features, including colony characteristics and the microscopic morphology of each isolate, such as vesicle type, phialide, and conidial arrangement (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Moreover, a fresh and pure culture of each isolate was prepared and stored at ambient temperature for subsequent investigations.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA extraction and molecular identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStrains were cultured on SDA (BioLife, Italy) and incubated at 30\u0026deg;C for 3 days. The genomic DNA of the strains was extracted via the method described by Makimura et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), with some modifications. A small amount of mycelia was crushed with a conical grinder in 500 \u0026micro;L of lysis buffer for 30\u0026ndash;60 s. Then, 150 \u0026micro;L of lithium acetate (CDH, New Delhi, India) was added, and the mixture was centrifuged twice at 12000 \u0026times; g for 3 min (at 4\u0026deg;C). The supernatant (DNA) was extracted by adding an equal volume (400\u0026ndash;500 \u0026micro;L) of isopropanol (Merck, Germany) at -20\u0026deg;C for 10 min and then centrifuging at 12000 \u0026times; g for 3 min (4\u0026deg;C). The isopropanol was discarded, and the DNA was washed with 300 \u0026micro;L of 70% ethanol at 12000 \u0026times; g for 10 min (at 4\u0026deg;C). Finally, the ethanol was discarded, and the DNA was dried and suspended in 50 \u0026micro;L of ultrapure water. The purified DNA was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until subsequent analysis. The partial sequence of the calmodulin gene (CAM) was amplified with the primers CMD5 (5-CCGAGTACAAGGAGGCCTTC-3) and CMD6 (5-CCGATAGAGGTCATAACGTGG-3) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSequence analysis and phylogenetic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe\u003c/b\u003e PCR products were subsequently sequenced with the forward calmodulin primer (CMD5) by the Cardio-Genetic Research Center sequence service (RCMRC, Tehran, Iran). The sequences were subsequently aligned via MEGA software (version 11), and the species were identified via searches of databases via the online BLAST system of the NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All sequences were submitted to DDBJ (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ddbj.nig.ac.jp\u003c/span\u003e\u003cspan address=\"https://www.ddbj.nig.ac.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and accession numbers were obtained. Phylogenetic relationships between \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e strains were constructed via the maximum likelihood method with MEGA software (version 11) and analysed to validate clade assignments.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the 629 clinical and environmental samples, 306 (48.65%) showed pure growth of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e species. Moreover, 323 (51.35%) yielded other fungi and some bacteria, which were not included in the study. Among the 261 clinical samples, only 131 isolates were identified, and among the 368 environmental samples, only 175 \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e isolates were identified (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Finally, 192 isolates (118 and 74 clinical and environmental isolates, respectively) were selected for sequence analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFrequencies of positive cultures from clinical and environmental samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSamples\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePositive\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eClinical (261)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOtomycosis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e141\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e110 (78.01%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBAL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e106\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19 (17.92%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSputum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (14.28%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eEnvironmental (368)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAir\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26 (23.63%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSoil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e258\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e149 (57.75%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e629\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e306 (48.65%)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSequencing of\u003c/b\u003e \u003cb\u003eAspergillus\u003c/b\u003e \u003cb\u003esection\u003c/b\u003e \u003cb\u003eNigri\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmong the 192 isolates of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e, 79 (41.15%) were identified as \u003cem\u003eA. tubingensis\u003c/em\u003e, followed by \u003cem\u003eA. welwitschiae\u003c/em\u003e (69, 35.94%), \u003cem\u003eA. niger\u003c/em\u003e (\u003cem\u003esensu stricto\u003c/em\u003e) (34, 17.71%), \u003cem\u003eA. neoniger\u003c/em\u003e (3, 1.56%), \u003cem\u003eA. piperis\u003c/em\u003e (3, 1.56%), \u003cem\u003eA. aculeatus\u003c/em\u003e (2, 1.04%), and \u003cem\u003eA. luchuensis\u003c/em\u003e (2, 1.04%). The accession numbers for the environmental and clinical isolates are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenBank accession numbers of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003enigri\u003c/em\u003e strains isolated from environmental and clinical samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEnvironmental isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eClinical isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. tubingensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC794827\u0026ndash;64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLC813269\u0026ndash;82\u003c/p\u003e\u003cp\u003eLC830486, LC830488, LC830492, LC830494 \u0026ndash; LC830495, LC830499, LC844950, LC844953, LC844955, LC844957\u0026ndash;8, LC844960\u0026ndash;1, LC844965, LC844967\u0026ndash;8, LC844970, LC844972\u0026ndash;4, LC844977, LC844979\u0026ndash;80, LC844984, LC844987\u0026ndash;8, LC844995\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. welwitschiae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC794865\u0026ndash;77,\u003c/p\u003e\u003cp\u003eLC794808\u0026ndash;811\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLC813244\u0026ndash;68, LC830485, LC830487, LC830489\u0026ndash;90, LC830493, LC830497, LC830500\u0026ndash;1, LC830503, LC844943, LC844947, LC844949, LC844951, LC844954, LC844959, LC844962\u0026ndash;64, LC844966, LC844969, LC844971, LC844981\u0026ndash;3, LC844985, LC844993, LC844996\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. niger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC794812\u0026ndash;25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLC813238\u0026ndash;43, LC844944\u0026ndash;46, LC844948, LC844956, LC844975\u0026ndash;6, LC844978, LC844986, LC844989\u0026ndash;92, LC844994\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. neoniger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC794806\u0026ndash;7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLC844952\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. piperis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC794826\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLC830491, LC830498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. aculeatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLC794804\u0026ndash;5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. luchuensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ndash;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLC830496, LC830502\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e74\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e118\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFrequency distribution of isolates in Iranian provinces\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe details of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e isolated from clinical and environmental samples are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Our results indicated that \u003cem\u003eA. welwitschiae\u003c/em\u003e was the most common isolate from clinical materials in Khuzestan, followed by \u003cem\u003eA. tubingensis\u003c/em\u003e, whereas \u003cem\u003eA. tubingensis\u003c/em\u003e was the most common strain, followed by \u003cem\u003eA. welwitschiae\u003c/em\u003e in Kohgiluyeh and Boyer-Ahmad (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, the environmental isolates shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e clearly indicate that \u003cem\u003eA. tubingensis\u003c/em\u003e was the most common environmental isolate of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e in all the sampled provinces except Esfahan.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetails of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e from clinical and environmental samples.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e Species\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eEnvironmental isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c8\" namest=\"c5\"\u003e\u003cp\u003eClinical isolates\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSoil\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAir\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOtomycosis\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSputum\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eBAL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. tubingensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. welwitschiae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. niger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. neoniger\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. piperis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. luchuensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eA. aculeatus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e118\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e192\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenic analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe maximum likelihood phylogenetic analysis of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e isolated from clinical and environmental samples is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. 79 \u003cem\u003eA. tubingensis\u003c/em\u003e, 3 \u003cem\u003eA. piperis\u003c/em\u003e, 3 \u003cem\u003eA. neoniger\u003c/em\u003e, 2 \u003cem\u003eA. aculeatus\u003c/em\u003e, and 2 \u003cem\u003eA. luchuensis\u003c/em\u003e strains representing clade 1. Clade 2 contained 2 subclades, A and B, with clade A containing 51 \u003cem\u003eA. welwitschiae\u003c/em\u003e and clade B containing 34 \u003cem\u003eA. niger\u003c/em\u003e and 18 \u003cem\u003eA. welwitschiae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUnderstanding the regional distribution of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e species and their antifungal susceptibility patterns is crucial for monitoring the emergence of antifungal resistance across various species. For several decades, more studies have shown that \u003cem\u003eA. niger\u003c/em\u003e is the predominant etiologic agent of otomycosis, with a worldwide distribution (\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). However, with respect to the exact differentiation of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e and the new taxonomy, new species were identified and presented. Our study revealed the frequency of clinical and environmental strains of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e in several Iranian provinces. \u003cem\u003eAspergillus welwitschiae\u003c/em\u003e (44.07%) was the most common strain isolated from clinical samples, followed by \u003cem\u003eA. tubingensis\u003c/em\u003e (34.75%) and \u003cem\u003eA. niger\u003c/em\u003e (16.95%). Similar studies from China (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) and France (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) have shown that \u003cem\u003eA. welwitschiae\u003c/em\u003e is the most common isolate from clinical samples, followed by \u003cem\u003eA. tubingensis\u003c/em\u003e and \u003cem\u003eA. niger\u003c/em\u003e. Moreover, Halvaeezadeh et al. obtained the same results that confirmed our results (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Importantly, \u003cem\u003eA. welwitschiae\u003c/em\u003e is closely related to \u003cem\u003eA. niger\u003c/em\u003e; thus, it may be misidentified as \u003cem\u003eA. niger\u003c/em\u003e via ITS region analysis (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Although \u003cem\u003eA. welwitschiae\u003c/em\u003e is the most common clinical isolate, \u003cem\u003eA. welwitschiae\u003c/em\u003e and \u003cem\u003eA. tubingensis\u003c/em\u003e are common clinical isolates from Khuzestan and Kohgiluyeh- Boyer-Ahmad, respectively.\u003c/p\u003e\u003cp\u003eIn recent years, several reports of chronic pulmonary aspergillosis or acute invasive aspergillosis caused by \u003cem\u003eA. welwitschiae\u003c/em\u003e have been published from Asian and Middle Eastern countries (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Recently, three cases of COVID-19-associated pulmonary aspergillosis (CAPA) caused by \u003cem\u003eA. welwitschiae\u003c/em\u003e were reported in immunocompetent patients in France (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and Brazil (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), and one case was reported in a Portuguese immunocompromised patient (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Najafzadeh et al. identified \u003cem\u003eAspergillus\u003c/em\u003e species (\u003cem\u003eA. tubingensis\u003c/em\u003e, \u003cem\u003eA. welwitschiae\u003c/em\u003e, \u003cem\u003eA. fumigatus\u003c/em\u003e, \u003cem\u003eA. sydowii\u003c/em\u003e, \u003cem\u003eA. neoniger\u003c/em\u003e, and \u003cem\u003eA. terreus\u003c/em\u003e) as the main causes of various infections, such as invasive aspergillosis (IA) in immunocompromised patients, and presented these infections with high mortality rates (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In another study, \u003cem\u003eA. welwitschiae\u003c/em\u003e was found to be the main cause of secondary pneumothorax in patients with a lung transplant. Fukushima et al. also demonstrated that this case highlights the potential of \u003cem\u003eA. welwitschiae\u003c/em\u003e to cause sudden pulmonary infection, presenting as pneumothorax, despite prophylactic treatment with itraconazole (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In a case study, \u003cem\u003eA tubingensis\u003c/em\u003e was identified as the cause of a solitary brain abscess in a Japanese man with alcoholic liver cirrhosis without any immunosuppressive disease (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Our study revealed that 38.9% of the ball isolates contained \u003cem\u003eA. welwitschiae\u003c/em\u003e. Notably, the presence of \u003cem\u003eA. welwitschiae\u003c/em\u003e usually indicates resistance to antifungal azole. According to previous studies, to the best of our knowledge, the initial resistance mechanism is attributed to the overexpression of the CYP51A gene, which is a major challenge for responding to treatment in patients (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In addition, unfortunately, \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e has been reported to have lower susceptibility to voriconazole (VOR), the first choice for treating invasive aspergillosis (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLike other \u003cem\u003eAspergillus\u003c/em\u003e species, Aspergillus section Nigri is a common saprophytic fungus in the environment. The environment plays an important role in the epidemiology of invasive aspergillosis. Several metrological conditions, as well as vegetation, play important roles in the mycoflora and distribution frequency in the environment. A study by Barberis et al. was conducted to identify the general correlation of the distribution of the \u003cem\u003eAspergillus\u003c/em\u003e group from Argentina on vineyard soils. In this study, \u003cem\u003eA. niger\u003c/em\u003e aggregates were the prevalent group (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). \u003cem\u003eAspergillus welwitschiae\u003c/em\u003e was isolated by Li et al. in China (2023) and identified as the causative agent of postharvest rot on Chinese cabbage (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In addition, the presence of three black aspergilli species, \u003cem\u003eA. tubingensis\u003c/em\u003e, \u003cem\u003eA. welwitschiae\u003c/em\u003e, and \u003cem\u003eA. ovarum\u003c/em\u003e, was reported in central Italy (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Moreover, \u003cem\u003eA. welwitschiae\u003c/em\u003e and \u003cem\u003eA. tubingensis\u003c/em\u003e are predominant in the soil, grapes, and air in the Vineyard, respectively (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe results of the phylogenetic analysis of the isolates revealed that the calmodulin gene is highly able to identify \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, clade 1 (clade \u003cem\u003eA. tubingensis\u003c/em\u003e) contained \u003cem\u003eA. tubingensis\u003c/em\u003e, \u003cem\u003eA. piperis\u003c/em\u003e, \u003cem\u003eA. neoniger\u003c/em\u003e, \u003cem\u003eA. aculeatus\u003c/em\u003e, and \u003cem\u003eA. luchuensis\u003c/em\u003e strains. Clade 2 (clade \u003cem\u003eA. niger\u003c/em\u003e aggregate) contained the closely related species \u003cem\u003eA. niger\u003c/em\u003e and \u003cem\u003eA. welwitschiae\u003c/em\u003e, which is similar to the results of Marques et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Moreover, Najafzadeh et al. phylogenetically analysed \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e and reported \u003cem\u003eA. niger\u003c/em\u003e and \u003cem\u003eA. welwitschiae\u003c/em\u003e in a clade that was completely separated from clade \u003cem\u003eA. tubingensis\u003c/em\u003e (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, knowledge of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e species distribution plays a crucial role in proper therapy. In summary, this study determined the presence of eight black aspergilli, \u003cem\u003eA. tubingensis\u003c/em\u003e, \u003cem\u003eA. welwitschiae\u003c/em\u003e, \u003cem\u003eA. niger\u003c/em\u003e, \u003cem\u003eA. neoniger\u003c/em\u003e, \u003cem\u003eA. piperis\u003c/em\u003e, \u003cem\u003eA. aculeatus\u003c/em\u003e, and \u003cem\u003eA. luchuensis\u003c/em\u003e, associated with clinical and environmental samples in Iran. As mentioned in the results, \u003cem\u003eA. tubingensis\u003c/em\u003e, \u003cem\u003eA\u003c/em\u003e. \u003cem\u003ewelwitschiae\u003c/em\u003e, and \u003cem\u003eA. niger\u003c/em\u003e were by far the predominant species. In terms of the source of isolation, \u003cem\u003eA. tubingensis\u003c/em\u003e was most common in the soil samples as well as in the air samples, \u003cem\u003eA. welwitschiae\u003c/em\u003e was most common in the otomycosis samples as well as in the BAL samples, and \u003cem\u003eA. tubingensis\u003c/em\u003e was most common in the sputum samples.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eA:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Aspergillus\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBAL:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eBronchoalveolar lavage\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFDA:\u003c/em\u003e\u003c/strong\u003e Food and Drug Administration\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSDA:\u003c/em\u003e\u003c/strong\u003e Sabouraud dextrose agar\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDNA:\u003c/em\u003e\u003c/strong\u003e Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePCR:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003ePolymerase chain reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ethics committee of Ahvaz Jundishapur University of Medical Sciences approved this project under reference no. IR.AJUMS.MEDICINE.REC.1402.020. The study was carried out in accordance with the Helsinki Rules. Additionally, written informed consent was obtained from all patients and parents (for children) involved in any experimental work with humans.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data have been included in the manuscript. However, raw data will be made available upon request from the corresponding author via email ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003cbr\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (grant number OG- 0208).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS. H. wrote the original draft, software, methodology, formal analysis, data curation; GA. J., SH. B., and M. K. resources, visiting patients, data curation; M. G.and H. J. Resources, preparing environmental samples; A. Z. M. writing, review \u0026amp; editing, supervision, project administration, data interpretation, funding acquisition, conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors express their gratitude to Jalal Hamzehee for collecting many of the study samples, as well as the bronchoscopy section and otolaryngology clinic of Imam-Khomeini Hospital, Ahvaz University of Medical Sciences, for the facilities provided to carry out the research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHashimoto K, Kawakami Y, Hashimoto R, Kitaoka Y, Onji Y, Oda H, et al. Distribution of Aspergillus section Nigri at shochu fermenting places in Japan. 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COVID-19-Associated Invasive Pulmonary Aspergillosis in the Intensive Care Unit: A Case Series in a Portuguese Hospital. J Fungi (Basel). 2021;7(10):881.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBenhadid-Brahmi Y, Hamane S, Soyer B, Mebazaa A, Alanio A, Chousterman B, et al. COVID-19-associated mixed mold infection: A case report of aspergillosis and mucormycosis and a literature review. J Mycol Med. 2022;32(1):101231.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCocio TA, Coelho VC, Del Negro GMB, Leite IGC, Gomes DCL, Xavier RSF, et al. First documentation of Aspergillus welwitschiae in COVID-19-associated pulmonary aspergillosis in the Americas. Rev Inst Med Trop Sao Paulo. 2025;67:e8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNajafzadeh MJ, Dolatabadi S, Zarrinfar H, Houbraken J. Molecular Diversity of Aspergilli in Two Iranian Hospitals. Mycopathologia. 2021;186(4):519\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFukushima S, Hagiya H, Ban S, Yaguchi T, Watanabe A, Tanaka S, et al. Secondary pneumothorax due to Aspergillus welwitschiae in a lung transplant recipient. Int J Infect Dis. 2025;154:107863.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eInaba T, Minami K, Ishioka H, Sekiguchi K, Watanabe A, Hatakeyama S. Isolated brain abscess caused by Aspergillus tubingensis in a patient with alcoholic liver cirrhosis. J Infect Chemother. 2025;31(5):102691.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarberis MG, Giaj Merlera G, Reynoso MM, Chulze SN, Torres AM. Factors affecting distribution and abundance of Aspergillus section Nigri in vineyard soils from grapevine growing regions of Argentina. J Sci Food Agric. 2014;94(14):3001\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, An Z, Yan L, Yaowei Z. Isolation, identification, and host range of Aspergillus welwitschiae causing postharvest rot on Chinese cabbage in China. Can J Plant Pathol. 2023;45(4):351\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCovarelli L, Tini F, Perrone G, Magist\u0026agrave; D, Onofri A, Beccari G. Survey on the occurrence of Aspergillus section Nigri species in grapes cultivated in Umbria (central Italy) and influence of several factors on their distribution. J Plant Pathol. 2025;107(1):229\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarques J, Castella G, Bragulat MR, Cabanes FJ. Diversity of Aspergillus section Nigri species from vineyards with different agro-climatic conditions in Catalonia, Spain. Int J Food Microbiol. 2025;430:111049.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus section Nigri, A. tubingensis, A. welwitschiae, Molecular identification, Calmodulin gene, Iran","lastPublishedDoi":"10.21203/rs.3.rs-6994062/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6994062/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground Aspergillus\u003c/h2\u003e\u003cp\u003esection \u003cem\u003eNigri\u003c/em\u003e is widely distributed in decaying plant materials in our environment. Although most of these species are known for food spoilage and some have industrial applications, some of these species, including \u003cem\u003eAspergillus tubingensis\u003c/em\u003e, \u003cem\u003eA. welwitschiae\u003c/em\u003e, and \u003cem\u003eA. niger\u003c/em\u003e, are etiologic agents of human and animal aspergillosis. Moreover, the frequency of each species in different environments is correlated with meteorological conditions. The present study aimed to investigate the frequency of \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e in clinical samples and environmental sources in several Iranian provinces.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eA total of 629 samples, including 261 clinical materials and 368 environmental materials, were collected from seven Iranian provinces. The \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri\u003c/em\u003e was initially screened on the basis of colony morphology and then subjected to sequencing via the calmodulin (\u003cem\u003eCaM\u003c/em\u003e) gene. Maximum likelihood phylogenies were constructed via MEGA 11 software.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOf the 192 \u003cem\u003eAspergillus\u003c/em\u003e section \u003cem\u003eNigri isolates\u003c/em\u003e, 41.15% were identified as \u003cem\u003eA. tubingensis\u003c/em\u003e, followed by \u003cem\u003eA. welwitschiae\u003c/em\u003e (35.94%), \u003cem\u003eA. niger\u003c/em\u003e (\u003cem\u003esensu stricto\u003c/em\u003e) (17.71%), \u003cem\u003eA. neoniger\u003c/em\u003e (1.56%), \u003cem\u003eA. piperis\u003c/em\u003e (1.56%), \u003cem\u003eA. aculeatus\u003c/em\u003e (1.04%), and \u003cem\u003eA. luchuensis\u003c/em\u003e (1.04%). The most prevalent species were \u003cem\u003eA. tubingensis\u003c/em\u003e and \u003cem\u003eA. welwitschiae\u003c/em\u003e from clinical and environmental samples, respectively. In addition, the frequency of these species varied across Iranian provinces.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study determined the presence and frequency, distribution, and phylogenetic relationships of eight black aspergilli, \u003cem\u003eA. tubingensis\u003c/em\u003e, \u003cem\u003eA. welwitschiae\u003c/em\u003e, \u003cem\u003eA. niger\u003c/em\u003e, \u003cem\u003eA. neoniger\u003c/em\u003e, \u003cem\u003eA. piperis\u003c/em\u003e, \u003cem\u003eA. aculeatus\u003c/em\u003e, and \u003cem\u003eA. luchuensis\u003c/em\u003e, associated with clinical and environmental samples from several Iranian provinces. In terms of the source of isolation, \u003cem\u003eA. tubingensis\u003c/em\u003e was most common in the soil and air samples, \u003cem\u003eA. welwitschiae\u003c/em\u003e was most common in the otomycosis and bronchoalveolar lavage (BAL) samples, and \u003cem\u003eA. tubingensis\u003c/em\u003e was most common in the sputum samples.\u003c/p\u003e","manuscriptTitle":"The frequency distribution of Aspergillus section Nigri from clinical and environmental samples in Iran","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 12:35:52","doi":"10.21203/rs.3.rs-6994062/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-31T05:33:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-28T19:45:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T20:42:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229044549132278913701177634831671627262","date":"2025-07-19T15:44:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"277789001109447893101251348639754633747","date":"2025-07-19T12:57:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T11:24:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330779563523867365302659385987254394277","date":"2025-07-16T05:52:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6657819569496342594004669485647494752","date":"2025-07-14T07:54:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101230310649475561298871284026013127363","date":"2025-07-14T06:19:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T05:13:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T04:38:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-09T04:35:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T15:12:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-07-07T18:05:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8f1d329-ad21-4a55-8076-9f12410e2d2f","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T16:01:17+00:00","versionOfRecord":{"articleIdentity":"rs-6994062","link":"https://doi.org/10.1186/s12866-025-04323-7","journal":{"identity":"bmc-microbiology","isVorOnly":false,"title":"BMC Microbiology"},"publishedOn":"2025-09-01 15:57:57","publishedOnDateReadable":"September 1st, 2025"},"versionCreatedAt":"2025-07-16 12:35:52","video":"","vorDoi":"10.1186/s12866-025-04323-7","vorDoiUrl":"https://doi.org/10.1186/s12866-025-04323-7","workflowStages":[]},"version":"v1","identity":"rs-6994062","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6994062","identity":"rs-6994062","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}