First Report of Iris domestica Leaf Spot Caused by Alternaria iridicola in China

Iris domestica, commonly known as “she gan”, is an herbaceous plant known for its medicinal properties and is distributed throughout China (Tang et al. 2025). In August 2025, leaf spot were observed on I. domestica plants in Handan (36.55°N, 113.26°E), Hebei Province, China (incidence rate 80%), plants in 150 ha were observed to be severely affected by the disease, causing a yield loss of 70%. Initial symptoms appeared on the leaves as circular yellow or brown lesions that expanded over time; later, the leaves turned yellow; and about 1 month later, the whole plant died. To isolate the causal agent, tissues (5×5 mm) from four symptomatic plants were removed from the border of lesions, surface sterilized in 75% ethanol for 30 s and 0.1% mercury chloride (HgCl2) for 1 min, then rinsed three times with sterile distilled water, plated on potato dextrose agar (PDA) at 25℃, and incubated in the dark for 7 days, two consistent surface morphology strains (202520 and 202521) were prepared based on single spore isolates. All isolates surface were gray-green and the reverse were dark green, the conidia were obclavate and light brown, 5 to 9 transverse and longitudinal septate, moderate in size (29.5 to 65.4 μm in length and 15.1 to 18.2 μm in width), the conidiophores were produced in clusters, erect or flexuous, according to the colony and conidia morphology, isolates were identified as Alternaria sp. (Woudenberg et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha (TEF gene), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and major allergen Alt a 1 (Alta1) regions were amplified (accession nos. PX588428 for ITS, PX549681 for tef1, PX549689 for GAPDH, and PX549690 for Alta1) with ITS1/ITS4 (Zhang et al. 2022), EF1-728F/EF1-986R (Glass and Donaldson 1995), GDF/GDR (Templeton et al. 1992), and Alt-F/Alt-R (Hong et al. 2005) primers. When compared with other Alternaria species in GenBank, the isolate exhibited 100% (ITS, LC269975), 100% (TEF-1α, OP374452), 100% (GAPDH, OP352299), and 100% (Alta1, OR486298) similarity with Alternaria iridicola (query coverage=100%, per-centage identity=100%). A phylogenetic tree was constructed in MEGA software (version 11.0.10) (Tamura et al. 2021) using the partial concatenated gene sequences. Neighbor-joining analysis revealed that the isolates were closely related to A. iridicola (100% bootstrap). Pathogenicity tests were performed on 5- to 6-month healthy seedlings of I. domestica, the culture disks of 202520 and 202521 was inoculated via leaf wounding on 36 plants per isolate. For the control treatment, 36 plants were treated with an equivalent area of PDA disks. The experiment was repeated three times. The plants were placed in a greenhouse from 26 to 30℃ and 95% relative humidity. The typical symptom were observed 10 days after inoculation, except on the control samples where no symptoms were observed. The same fungus was successfully reisolated from the symptomatic plant tissue and was reidentified as A. iridicola through morphological characteristics, ITS, TEF, GAPDH and Alta1 sequencing analysis (accession nos. ITS: PX925249, TEF: PX926523, GAPDH: PX926521 and Alta1: PX926522), thereby fulfilling Koch’s postulates. This study represents the first documented occurrence of A. iridicola induced leaf spot on I. domestica in China. The finding is greatly significant for preventing and controlling root rot in I. domestica and reducing economic losses for farmers.