Identification and phylogenetic placement of the fungal isolate S14 from Silybum marianum within Aspergillus section Nigri

Silybum marianum o‘simligidan ajratib olingan endofit zamburug‘ izolyati S14 340 bp uzunlikdagi ITS ketma-ketligi yordamida tekshirildi va Aspergillus section Nigri tipik shtammlarining referens ketma-ketliklari bilan solishtirildi. Filogenetik tahlil S14 ni section Nigri tarkibiga joylashtirdi; beshta yaqin qarindosh tipik shtamm bilan ketma-ketlik identikligi 98,5% ni tashkil etdi. Tahlil qilingan ITS sohasi bu turlarni bir-biridan ajratmagani uchun izolyat tasdiqlovchi sekvenatsiya va qo‘shimcha lokuslar tahlili olinmaguncha Aspergillus sp. (section Nigri) S14 deb nomlandi.

FUNDAMENTAL VA AMALIY MIKROBIOLOGIYANING BUGUNGI HOLATI VA RIVOJLANISH ISTIQBOLLARI: YOSH OLIMLAR NIGOHI Respublika ilmiy-amaliy anjumani · Toshkent, 2026-yil 30-oktabr

II BIOTEXNOLOGIYA, MOLEKULYAR BIOLOGIYA VA BIOINFORMATIKA DOI 10.66073/ReFocus-Conf/3010056

IDENTIFICATION AND PHYLOGENETIC PLACEMENT OF THE FUNGAL ISOLATE S14 FROM SILYBUM MARIANUM WITHIN ASPERGILLUS SECTION NIGRI Khayrullaeva Lobar Mansur qizi¹, Umarova Gulbakhor¹, Eshboev Farhod Bakir o‘g‘li²* ¹ Department of Biochemistry, National University of Uzbekistan, 100174 Tashkent, Uzbekistan ² Institute for Advanced Studies at New Uzbekistan University, 100007 Tashkent, Uzbekistan * Corresponding author: f.eshboev@newuu.uz Abstract: Endophytic fungal isolate S14 from Silybum marianum was examined using a 340-bp ITS sequence and compared with reference sequences of Aspergillus section Nigri type strains. Phylogenetic analysis placed S14 within section Nigri, with 98.5% sequence identity to five closely related type strains. Because the analyzed ITS region did not discriminate among these species, the isolate was designated Aspergillus sp. (section Nigri) S14 pending confirmatory sequencing and analysis of additional loci. Keywords: Silybum marianum; endophytic fungi; Aspergillus section Nigri; ITS sequencing; phylogenetic analysis; fungal identification. Introduction Endophytic fungi inhabit healthy plant tissues without causing visible symptoms and are an established source of structurally diverse secondary metabolites. Medicinal plants such as Silybum marianum (milk thistle), known for silymarin, are attractive hosts for bioprospecting. Reliable isolate identification is a prerequisite for such investigations. The internal transcribed spacer (ITS) region is the standard first-line fungal DNA barcode. This study aimed to identify isolate S14 recovered from S. marianum using ITS sequence analysis and to assess whether the available barcode supports species-level identification. Materials and Methods A single ITS4-primed sequence read of 340 bp, spanning the 5.8S rRNA gene and adjacent ITS regions, was obtained from isolate S14. The sequence was compared with six Aspergillus section Nigri type-strain sequences from the NCBI RefSeq targeted-loci database: A. niger (NR_111348.1), A. tubingensis (NR_131293.1), A. luchuensis (NR_135449.1), A. costaricensis (NR_103604.1), A. foetidus (NR_163668.1), and A. hubkae (NR_200571.1). Aspergillus flavus (NR_111041.1; section Flavi) served as an outgroup. Phylogenetic relationships were visualized using a UPGMA tree. Results Isolate S14 clustered within Aspergillus section Nigri (Figure 1). It differed from the type strains of A. niger, A. tubingensis, A. luchuensis, A. costaricensis, and A. foetidus at five of 341 alignment positions (98.5% identity), and from A. hubkae at ten of 343 positions (97.1% identity). Divergence from the A. flavus outgroup was 7.5%. The five closest reference sequences were identical throughout the analyzed window, whereas A. hubkae differed from them at five positions.

FOR CITATION Khayrullaeva L., Umarova G., Eshboev F. Identification and phylogenetic placement of the fungal isolate S14 from Silybum marianum within Aspergillus section Nigri // Proceedings of the Republican Scientific and Practical Conference “Status and Development Prospects of Fundamental and Applied Microbiology: The Viewpoint of Young Scientists”. – Tashkent, 2026. – P. 205–206. DOI: https://doi.org/10.66073/ReFocus-Conf/3010056

FUNDAMENTAL VA AMALIY MIKROBIOLOGIYANING BUGUNGI HOLATI VA RIVOJLANISH ISTIQBOLLARI: YOSH OLIMLAR NIGOHI 30.10.2026

Discussion The short ITS window cannot distinguish the five closest species. All five observed differences between S14 and its nearest references occurred in G/C-rich tracts, consisting of three single-nucleotide insertions or deletions and one adjacent GC/CG exchange. These differences may arise from base-calling uncertainty in a single sequence read and should not be interpreted as species-specific markers without sequencing the opposite strand. Further analysis of calmodulin and β-tubulin loci is needed to resolve the isolate at species level.

Figure 1. UPGMA tree of the ITS region (340-bp window) placing Aspergillus sp. S14 (red) among Aspergillus section Nigri type strains; A. flavus (section Flavi) is the outgroup. Scale bar: 0.01 substitutions per site Conclusion The available ITS sequence supports assignment of isolate S14 to Aspergillus section Nigri but does not permit reliable species-level identification. Until complementary sequence data and additional genetic markers become available, the isolate should be designated Aspergillus sp. (section Nigri) S14.