Identification and phylogenetic placement of an endophytic Alternaria isolate D15 from Ferula khustanica

Ferula khustanica (Apiaceae) o‘simligidan ajratib olingan endofit zamburug‘ izolyati D15 ning taksonomik o‘rnini belgilash uchun ichki transkribatsiyalanuvchi spacer (ITS) sekvenatsiyasi qo‘llanildi. Sanger usulida olingan 582 nukleotidli teskari zanjir o‘qilishi beshta Alternaria tipik shtammining referens ketma-ketliklari bilan solishtirildi va UPGMA usulida filogenetik daraxt qurildi. Izolyat Alternaria avlodi ichida klasterlandi va tekshirilgan tipik shtammlar bilan ketma-ketlik identikligi 99,2–99,8% ni tashkil etdi. Biroq bu referens turlar o‘rtasida ITS tafovuti past bo‘lgani uchun turni ishonchli belgilash imkonini bermaydi. Shu sababli izolyat multilokusli tavsif olinmaguncha Alternaria sp. D15 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/3010055

IDENTIFICATION AND PHYLOGENETIC PLACEMENT OF AN ENDOPHYTIC ALTERNARIA ISOLATE D15 FROM FERULA KHUSTANICA Khamdamov Doniyor Shuxratovich¹ and Eshboev Farhod Bakir o‘g‘li¹* ¹ Institute for Advanced Studies at New Uzbekistan University, 100007 Tashkent, Uzbekistan * Corresponding author: f.eshboev@newuu.uz Abstract: An endophytic fungal isolate, D15, recovered from Ferula khustanica (Apiaceae), was investigated to establish its taxonomic placement using internal transcribed spacer (ITS) sequencing. A 582-base reverse-strand Sanger read was compared with reference sequences from five Alternaria type strains, and a UPGMA phylogenetic tree was constructed. The isolate clustered within Alternaria and showed 99.2–99.8% sequence identity to the examined type strains. However, the low ITS divergence among these reference species prevents a reliable species-level assignment. The isolate is therefore designated Alternaria sp. D15 pending multilocus characterization. Keywords: Alternaria; Ferula khustanica; endophytic fungi; ITS sequencing; molecular identification; phylogenetic analysis. Introduction Endophytic fungi colonize internal plant tissues and may represent valuable sources of structurally diverse secondary metabolites. Accurate taxonomic identification is essential before their biological and biosynthetic potential can be evaluated. The internal transcribed spacer (ITS) region is widely used as a fungal DNA barcode, although its resolution varies among taxonomic groups. This study aimed to determine the phylogenetic position of fungal isolate D15 obtained from Ferula khustanica and to assess whether the available ITS sequence supports identification at the species level. Materials and Methods The ITS region of isolate D15 was analyzed using a 582-base reverse-strand Sanger sequencing read spanning the ITS4–ITS5 region. The sequence was compared by semi-global alignment with ITS sequences of five Alternaria type strains retrieved from the NCBI RefSeq targeted-loci database: A. destruens (NR_137143.1), A. alstroemeriae (NR_163686.1), A. doliconidium (NR_158361.1), A. tectorum (NR_187081.1), and A. eichhorniae (NR_111832.1). Pseudophaeosphaeria rubi (NR_154351.1) was used as an outgroup. A UPGMA tree was reconstructed using pairwise p-distances. Results Phylogenetic analysis placed isolate D15 within the genus Alternaria (Figure 1). Its ITS sequence differed from A. destruens at 1 of 521 aligned positions (99.8% identity), from A. alstroemeriae and A. doliconidium at 2 of 530 and 2 of 529 positions, respectively (99.6%), from A. tectorum at 3 of 520 positions (99.4%), and from A. eichhorniae at 4 of 521 positions (99.2%). The outgroup exhibited 17.6% sequence divergence. The five Alternaria type strains differed from one another by only 0.2–0.8% in the analyzed region.

FOR CITATION Khamdamov D., Eshboev F. Identification and phylogenetic placement of an endophytic Alternaria isolate D15 from Ferula khustanica // 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. 203–204. DOI: https://doi.org/10.66073/ReFocus-Conf/3010055

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

Discussion The close similarity of the examined ITS sequences supports assignment of D15 to Alternaria but does not allow confident discrimination among the reference species. Although A. destruens showed the highest sequence identity, this observation alone is insufficient to identify D15 as that species. Additional sequence data from more informative loci, such as GAPDH, RPB2, and TEF1, are needed for a robust species-level determination. Conclusion ITS sequencing and phylogenetic analysis identified D15 as a member of the genus Alternaria. Given the limited discriminatory power of the available ITS region, the isolate should be reported as Alternaria sp. D15 until multilocus analysis provides a more precise taxonomic assignment.

Figure 1. UPGMA tree based on ITS sequences showing the placement of Alternaria sp. D15 (red) relative to five Alternaria type strains, with Pseudophaeosphaeria rubi as the outgroup. Basal branches are truncated (//)