First Report of <i>Pseudomonas graminis</i> Causing Bacterial Leaf Streak of <i>Allium microdictyon</i> in China

Allium microdictyon, a perennial herb thriving in temperate mountain forests of the Northern Hemisphere, serves as a valuable economic resource with dual culinary and medicinal applications. In May 2024, a severe bacterial infection affecting A. microdictyon was identified in Qinghe Town, Heilongjiang Province, China (46.18°N, 129.14°E), with infection rates reaching 10% - 15% of local plant populations. The pathogen primarily infects the leaves of A. microdictyon, initially manifesting as pale green linear lesions on the leaf surface that gradually extend along the longitudinal axis of the leaf. As the disease progresses, the infected tissue develops distinct yellow streak-like lesions. Based on these characteristic symptoms, we named this disease bacterial leaf streak of A. microdictyon. To isolate and identify the causative pathogen, diseased tissues were surface-disinfected in 75% ethanol for 30 seconds, rinsed three times with sterile water, then ground in a sterile mortar. The extract was plated on nutrient agar (NA) and incubated at 28°C for 48h. Three similar colonies (AM01, AM02, AM03) were isolated. After transfer to NA and incubation at 28°C for 72 hours, the colonies exhibited circular morphology with smooth, moist edges, yellow, slight elevation, and diameters ranging from 2 to 3 mm. The strains were identified as aerobic, Gram-negative, rod-shaped bacteria, with cell dimensions of 1.92±0.2 µm in length and 0.69±0.08 µm in width (n = 50). The 16S rRNA genes were amplified and sequenced to confirm that three isolates belonged to the genus Pseudomonas. Multilocus sequence analysis (MLSA) was performed on the four housekeeping genes (16S rRNA, dnaK, gyrB, and rpoD) of the isolates, and the sequences were submitted to GenBank. Accession numbers for these genes from strains AM01, AM02, and AM03 are as follows: AM01 (PV355175, PV366778, PV366779, PV366780), AM02 (PX279508, PX290093, PX290094, PX290096), AM03 (PX279509, PX290092, PX290095, PX290097). Aligned concatenated sequences were used to construct a maximum likelihood phylogenetic tree in MEGA12, revealing clustering with Pseudomonas graminis PgKB30 (GenBank: CP053746.1), with 98.7%-99.2% nucleotide similarity. Pathogenicity was confirmed through bacterial suspension (OD600=1.0) infiltration into 4-week-old plants at room temperature. Following inoculation, the plants were maintained in growth chambers under controlled environmental conditions(≥25℃, ≥90% RH) . Plants inoculated with sterile water were used as the negative control. Distinct leaf streak symptoms developed on plants inoculated with three isolates within 7 days, while controls remained asymptomatic. Bacteria were reisolated from diseased tissues, and Gram staining and 16S rRNA gene sequencing confirmed that the reisolated bacteria matched the original pathogen. All experiments were repeated three times, yielding consistent results, fully satisfying Koch’s postulates. This is first report of P. graminis causing bacterial leaf streak in A. microdictyon. The emergence of this disease has adversely affected the commercial value of A. microdictyon, thereby necessitating close attention from relevant industry stakeholders.