ITS-based identification and phylogenetic characterization of endophytic Alternaria sp. H8 isolated from Helichrysum mussae

Helichrysum mussae o‘simligidan ajratib olingan endofit zamburug‘ izolyati H8 ning taksonomik o‘rnini aniqlashtirish uchun ichki transkribatsiyalanuvchi spacer (ITS) ketma-ketligi tahlil qilindi. Sifati bo‘yicha kesib olingan 544 bp uzunlikdagi teskari zanjir ITS o‘qilishi beshta Alternaria tipik shtammining referens ketma-ketliklari bilan solishtirildi. Filogenetik tahlil H8 izolyatini Alternaria avlodi tarkibiga joylashtirdi. Izolyat A. destruens tipik shtammi bilan tekislangan 521 pozitsiyada mos keldi, yana to‘rtta tur bilan ketma-ketlik identikligi 99,4–99,8% ni tashkil etdi. ITS sohasi referens taksonlar o‘rtasida kam o‘zgaruvchanlik ko‘rsatgani uchun bu natijalar avlod darajasidagi identifikatsiyani tasdiqlaydi, ammo turni aniq belgilashga asos bermaydi. Tur darajasida aniqlash uchun qo‘shimcha multilokusli sekvenatsiya talab etiladi.

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/3010053

ITS-BASED IDENTIFICATION AND PHYLOGENETIC CHARACTERIZATION OF ENDOPHYTIC ALTERNARIA SP. H8 ISOLATED FROM HELICHRYSUM MUSSAE Ubaydullaev Ziyoxo‘ja¹, Khujanov Alisher², Fayziev Voxid Baxramovich¹, Madaminov Sulaymon³, Eshboev Farhod Bakir o‘g‘li³* ¹ Chirchik State Pedagogical University, Chirchik 111720, Tashkent Region, Uzbekistan ² Samarkand State University, Samarkand 140104, Uzbekistan ³ Institute for Advanced Studies at New Uzbekistan University, Tashkent 100007, Uzbekistan * Corresponding author: f.eshboev@newuu.uz Abstract: An endophytic fungal isolate, H8, recovered from Helichrysum mussae, was investigated using internal transcribed spacer (ITS) sequence analysis to clarify its taxonomic placement. A quality-trimmed, 544-bp reverse-strand ITS read was compared with reference sequences of five Alternaria type strains. Phylogenetic analysis placed H8 within the genus Alternaria. The isolate matched the A. destruens type strain across 521 aligned positions and shared 99.4–99.8% sequence identity with four additional species. Because the ITS region showed limited variation among the reference taxa, these findings support identification at the genus level but do not establish a definitive species assignment. Additional multilocus sequencing is required for species-level resolution. Keywords: endophytic fungi; Helichrysum mussae; Alternaria; ITS sequencing; fungal identification; phylogenetic analysis. Introduction Endophytic fungi colonize internal tissues of apparently healthy plants and represent a valuable resource for exploring fungal diversity and specialized metabolism. Accurate taxonomic characterization is essential before assessing their biological activities or biotechnological applications. Although the internal transcribed spacer (ITS) region is widely used as a fungal DNA barcode, closely related species may share highly similar ITS sequences. This study aimed to determine the phylogenetic position of fungal isolate H8 obtained from Helichrysum mussae and to evaluate the taxonomic resolution provided by its ITS sequence. Materials and Methods A single reverse-strand ITS sequence of 544 bp was obtained from isolate H8 after quality trimming (mean Phred score: 34). The sequence was aligned with ITS reference sequences from five Alternaria type strains: 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) served as the outgroup. Four ambiguous nucleotide calls were treated as compatible with their corresponding aligned reference bases. Sequence similarity and phylogenetic placement were evaluated using the aligned ITS region. Results The ITS-based phylogenetic analysis positioned isolate H8 within Alternaria (Figure 1). The sequence was identical to that of the A. destruens type strain across 521 aligned positions. Comparisons with A. alstroemeriae and A. doliconidium each revealed one difference (99.8%

FOR CITATION Ubaydullaev Z., Khujanov A., Fayziev V., Madaminov S. et al. ITS-based identification and phylogenetic characterization of endophytic Alternaria sp. H8 isolated from Helichrysum mussae // 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. 198–199. DOI: https://doi.org/10.66073/ReFocus-Conf/3010053

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

identity), whereas A. tectorum and A. eichhorniae differed at two (99.6%) and three (99.4%) positions, respectively. The outgroup showed 17.9% sequence divergence. Discussion The close ITS similarity between H8 and multiple Alternaria type strains indicates that this marker alone cannot reliably discriminate among the examined species. Differences relative to A. alstroemeriae, A. doliconidium, and A. tectorum involved single-base length variants in adeninerich regions, while A. eichhorniae showed two additional substitutions. Consequently, the proximity of H8 to A. destruens in the phylogenetic tree should not be interpreted as conclusive evidence of species identity. Analysis of additional loci, including GAPDH, RPB2, and TEF1, would provide greater taxonomic resolution.

Figure 1. ITS-based phylogenetic tree showing the placement of Alternaria sp. H8 (red) among Alternaria type strains, with Pseudophaeosphaeria rubi as the outgroup. Truncated basal branches are indicated by //. Scale bar: 0.002 substitutions per site Conclusion ITS sequence comparisons support assignment of the endophytic isolate H8 to the genus Alternaria. However, the limited sequence divergence among closely related reference species prevents confident species-level identification. The isolate should therefore be designated Alternaria sp. H8 pending multilocus confirmation.