First Report of <i>Polygonatum kingianum</i> Leaf Spot Disease Caused by <i>Alternaria alstroemeriae</i> in Honghe Prefecture, Yunnan, China

Polygonatum kingianum Collett &amp; Hemsl., a perennial herbaceous plant native to China, is widely distributed in Yunnan Province (Wu et al. 2024). In July 2025, a severe outbreak of leaf blight was observed on P. kingianum at a cultivation base in Pingbian Miao Autonomous County, Honghe Prefecture, Yunnan Province, China. The affected cultivation area spanned approximately 1.5 ha, with a disease incidence of 100% leading to an estimated yield reduction of 40-50%. Infected leaves exhibited small brown lesions with dehydrated centers. These lesions were characterized by brown centers and yellow margins. In severe cases, the lesions coalesced, causing leaf desiccation and premature abscission. For pathogen isolation, symptomatic leaves were randomly sampled from the upper, middle, and lower canopy of twenty individual plants, yielding a total of twenty leaves. The leaf tissues were surface-sanitized by sequential immersion in 75% ethanol for 1 min and 0.1% mercuric chloride for 1 min, followed by five rinses with sterile water. After drying, the tissue fragments were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 5 days. Morphologically uniform fungi were isolated from all twenty symptomatic leaf samples, resulting in a 100% isolation rate. Four isolates were randomly selected for purification and preservation. Subsequently, a representative isolate was chosen for further study and designated as strain PKD-1. The colonies on PDA were brown. The conidia were brown and obclavate, measuring 29 μm × 15 μm (n = 50). Each conidium typically produced an elongated, aseptate or sparsely septate, beak-like apical cell. The total length of the conidium, including the beak, reached up to 120 μm. Based on morphological and microscopic characteristics, the isolated strain showed a high degree of similarity to an Alternaria sp. reported by Lawrence et al. (2016). The genomic DNA of our isolate was extracted using the Ezup Column Fungi Genomic DNA Purification Kit. The internal transcribed spacer (ITS), large ribosomal subunit (LSU), and translation elongation factor 1-alpha (tef1) genes were amplified via polymerase chain reaction using the primer pairs ITS1/ITS4 (Li et al. 2025), LSU (Farha et al. 2019), and tef1 (Woudenberg et al. 2015), respectively. The sequences were deposited in public repositories as follows: the ITS, LSU and tef1 sequences in GenBank under accession numbers PX519198, PX519308 and PX757113, respectively. BLASTN analysis of the ITS, LSU and tef1 gene sequences showed high homology with existing deposits: the ITS (546/547 bp) was 99.82% identical to NR_163686.1; the LSU (842/843 bp) was 99.88% identical to NG_069882.1; the tef1 (236/240 bp) was 98.33% identical to KP125072.1. Phylogenetic analysis based on ITS, LSU and tef1 sequences revealed that strain PKD-1 clustered within the same clade as the type strain CBS 118809 of A. alstroemeriae. Based on morphological characteristics, pathogenicity tests, and multi-locus phylogenetic analysis of the ITS and LSU genes, the pathogen causing leaf spot on P. kingianum was identified as A. alstroemeriae. A pathogenicity test was performed three times to fulfill Koch's postulates on one-year-old P. kingianum plants. For each test, nine potted replicates per treatment were used, and the assessments were conducted on the leaves. Inoculation with 50 µl of a 105 spores/mL conidial suspension induced characteristic brown spots on leaves after 5 days at 18-25℃, whereas controls treated with sterile water remained asymptomatic. The pathogen was re-isolated from symptomatic tissue and confirmed as A. alstroemeriae through morphological comparison and phylogenetic analysis of the ITS, LSU, and tef1 gene sequences. To our knowledge, this is the first report of A. alstroemeriae infecting P. kingianum in China. This finding identifies a new disease threat to this valuable medicinal crop and provides crucial etiological information for developing targeted management strategies.