First Report of Bacterial Leaf Spot on Marigold ( <i>Tagetes erecta</i> L.) Caused by <i>Pseudomonas cichorii</i> in Yunnan Province, China

Marigold (Tagetes erecta L.), a major economic crop as a material of lutein, is widely cultivated across the world. In August 2025, leaf spot symptoms were observed on approximately 533 ha of marigold fields in Tengchong County (24.98˚N, 98.40˚E), Yunnan Province, China. Initially, small, dark brown, irregular spots with distinct chlorotic halos appeared on older leaves. These spots gradually enlarged and coalesced to form large necrotic areas. Subsequently, similar spots newly appeared on tender leaves. To isolate and identify the causal pathogen, typical symptomatic leaves were collected and cut into 5 × 5 mm sections. These leaf tissues were sterilized in 75% ethanol for 5 s and 1% sodium hypochlorite for 3 mins, then rinsed four times with sterile water. The dried tissues were plated onto nutrient agar (NA) and incubated at 28℃ for 48h. White, round colonies with smooth margins were isolated and all of them were Gram-negative. Four strains (SQ2, SQ4, SQ7 and SQ8) were selected for molecular identification and pathogenicity tests. Genomic DNA of four strains were extracted and 16s rRNA was amplified with universal primers 27F/1492R (Frank et al. 2008). BLAST analysis revealed that the sequences (GenBank Accession nos. PX664326-PX664329) shared 99% similarity with those of Pseudomonas cichorii. Three housekeeping genes (gyrB, rpoB and rpoD) were amplified with primers UP1E/APrU, LAPS5/LAPS27 and rpoD-F/rpoD-R (Mulet et al. 2010; Sarkar et al. 2004), and the sequences were submitted to GenBank (GenBank Accession Nos. gyrB: PX667009-PX667012; rpoB: PX667013-PX667016; rpoD: PX667017-PX667020). A phylogenetic tree was constructed using the maximum likelihood method based on multilocus sequence analysis (MLSA) by IQ-tree. MLSA indicated that all four strains clustered with P. cichorii PPST50936 and P. cichorii DSM50259 with support rate 100%. To fulfill Koch's postulates, bacterial suspension (OD600=0.6) was sprayed on needle punctured leaves of 6-weeks-old marigold at greenhouse. LB liquid medium was treated as control group. Five days after inoculation, leaf spot symptoms similar to those in the field were observed, while the control showed no symptoms. P. cichorii was re-isolated from inoculated leaves but not from controls, fulfilling Koch’s postulates. The re-isolated bacteria were confirmed based on morphology and DNA sequence. Tabacco leaf spot caused by P. cichorii have been reported in China (Lan et al. 2022). To our knowledge, this is the first report of P. cichorii causing leaf spot on Marigold (T. erecta L.) in China. This study will lay a theoretical foundation for the prevention and control of this disease.