First Report of Leaf Rot on Chinese Chive ( Allium tuberosum Rottler ex Spreng.) Caused by Phytophthora heterospora in Yunnan Province, China

Chinese chives (Allium tuberosum Rottler ex Spreng.), an economically important aromatic vegetable, are widely cultivated worldwide. In May 2025, a severe leaf rot disease was observed on Chinese chives in Mile (24.45°N, 103.4°E), Yunnan Province, China. Following consecutive days of rain and high humidity, symptoms first appeared on young leaves. These symptoms manifested as progressive necrotic rot that extended from the middle section toward both leaf apices and bases. Field surveys revealed that over 75% of surveyed plants exhibited typical rot symptoms. To identify the causal agent, we collected 10 diseased plant samples. After surface cleaning, the samples were cut into 5 × 5 mm tissue segments. These segments were then surface-sterilized with 75% ethanol for 1 min, and rinsed three times with sterile distilled water. The tissues were transferred to potato dextrose agar (PDA) medium and incubated at 25 °C in the dark for 3 days. Following purification of the putative pathogen, ten isolates displaying consistent morphological characteristics were obtained. One of these isolates, designated as JC9, was selected for further characterization. Isolate JC9 cultured on PDA at 25 °C for 5 days produced abundant non-septate aerial hyphae. Pseudoconidia were spherical to ovoid in shape. Sporangia displayed morphological heterogeneity, encompassing ovoid forms with laterally positioned papillae and limoniform forms bearing short pedicels possessing prominent papillae. The mean sporangial length and width were 55.1 μm and 25.5 μm, respectively. Occasionally, empty sporangia exhibiting direct apical germination were observed. Chlamydospores were globose, occurring either terminally or intercalarily, with a mean diameter of approximately 28 μm. Collectively, these morphological features align closely with the established diagnostic criteria for Phytophthora heterospora (Scanu et al. 2021). For molecular identification, three gene regions were selected for amplification: the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1), and beta-tubulin (β-TUB). These markers are widely adopted for Phytophthora species identification and phylogenetic analysis, and are recommended for multi-locus phylogenies (Kroon et al. 2004). Amplifications were performed using the primer pairs ITS4/ITS5 (White et al. 1990), ELONGF1/ELONGR1 and F-βtub1/F-βtub2r (Kroon et al. 2004), respectively. The sequences were deposited in GenBank under accession numbers PZ266852 (ITS), PZ242888 (TEF1), and PZ242889 (β-TUB). Phylogenetic analyses — conducted under both maximum likelihood and Bayesian inference frameworks and based on concatenated alignments of the three loci — demonstrated that isolate JC9 formed a highly supported monophyletic clade (100% bootstrap support; posterior probability = 1.0) with reference strains of Phytophthora heterospora within the Phytophthora palmivora species complex. This clade was phylogenetically distinct from the Phytophthora palmivora sensu stricto lineage, thereby confirming the taxonomic assignment of isolate JC9 to Phytophthora heterospora. Pathogenicity tests were conducted by inoculating individual leaves of potted Chinese chive ( Allium tuberosum) plants with 5 µL zoospore suspension (1 × 10⁶ spores/mL) of JC9. The zoospores were harvested from PDA cultures using sterile water. Control leaves were treated with an equal volume of sterile distilled water. Each treatment comprised 30 healthy leaves with three independent biological replicates. All plants were maintained in a controlled greenhouse at 28 ℃ under a 12-h photoperiod (12-h light / 12-h dark). Sterile water was sprayed once daily to maintain persistent high humidity for pathogen infection. Three days post-inoculation, lesions identical to field symptoms developed on inoculated leaves, whereas control leaves remained asymptomatic. A pathogen strain morphologically and molecularly identical to isolate JC9 was successfully re-isolated from symptomatic leaf tissue, confirming Koch's postulates. Leaf rot of Chinese chives (Allium tuberosum) caused by Phytophthora heterospora has recently become a serious disease during the rainy season in Mile, significantly affecting local production and causing economic losses. As a major Chinese chive production area in southwest China, Mile features a subtropical monsoon climate characterized by high temperatures, abundant rainfall, and prolonged soil wetness during the rainy season — environmental conditions highly favorable for Phytophthora heterospora, a fungus-like oomycete pathogen known to thrive in warm, humid environments with distinct wet periods. These climatic and edaphic factors collectively facilitate the pathogen’s infection, colonization, and dissemination in chive fields. To date, this disease has not been reported in other Chinese chive-growing regions. Primary transmission pathways include infected seedlings and pathogen-contaminated soil; under field conditions, rain splash serves as the dominant mechanism for short-distance dispersal. This study provides a fundamental basis for early diagnosis, rapid detection, and integrated management of this emerging leaf rot disease.