Tobacco (Nicotiana tabacum L.) is an economically important crop in China. In April 2024, field tobacco (cv. Yueyan97) showed wilting with leaf chlorosis in Nanxiong of Guangdong Province. Diseased roots had brown to black lesions along the length of tap root, accompanied by decay and sloughing of the root cortex of lateral roots. Disease incidence ranged from 10 to 32% in the surveyed fields, 60 ha in total. Five symptomatic plants with typical leaf chlorosis and root rot were randomly collected from the surveyed fields for pathogen isolation. Root segments (0.5 cm) were surface sterilized in 75% ethanol for 30 s followed by rinsing with sterile distilled water three times. Twenty-five air dried root pieces were placed on potato dextrose agar (PDA, amended with 100 mg L-1 streptomycin sulphate) and incubated at 25℃ in the dark for 2 days. Nine pure isolates were obtained, four isolates had typical morphological characteristics of Fusarium oxysporum, and the other five isolates (Nx01 to Nx05) showed abundant fluffy, white to cream aerial mycelia with a hyaline ring on the surface, and an average growth rate of 15.25±0.37 mm/day on PDA. The hyphae were coenocytic with no septa. Thick-walled oospores were measuring 23.84±3.56 μm. The morphological characteristics were consistent with the Aphanomyces species (Papabizas and Ayers 1974). All nine isolates were tested for pathogenicity to fulfill the Koch's postulates. Four-leaf stage healthy tobacco seedlings (Yueyan97, n=30) were inoculated by pouring 10 mL zoospore suspension (104 zoospores mL-1) around the rhizosphere for Nx01-Nx05 (Kumar et al., 2021), and 10 mL conidial suspensions (1.0× 105 cfu/ml) for F. oxysporum. Control seedlings were inoculated with sterilized water (n=30). All the treatments were maintained under greenhouse conditions with a 12-h light/dark photoperiod at 25±0.5℃ and 70%RH for 30 days. The assay was conducted three times. Foliage chlorosis and root rot were observed on the inoculated seedlings. The disease incidence was 100% in the Nx01 to Nx05 treatments, and 30% in the F. oxysporum treatments. The control seedlings remained asymptomatic after 30 days. The pathogens were reisolated from the inoculated seedlings and were identified by morphological characteristics. F. oxysporum is a known pathogen of tobacco root rot. This result showed that isolates Nx01 to Nx05 can cause root rot of tobacco. The total DNA of Nx01 to Nx05 was extracted from mycelium of the 5-day-old cultures by CTAB method (White et al. 1990). The ribosomal DNA internal transcribed spacer (ITS) region, cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2), and the beta-tubulin gene were amplified using primer pairs ITS5/ITS4, Oom-COI-Lev-up/FM85-mod, Cox2-F/FMPhy-10b and Btub_F1A/Btub_R1A respectively, and sequenced (Kageyama et al. 2023, Robideau et al. 2011, White et al. 1990). Maximum likelihood analysis was carried out using MEGA 11. Sequences of Nx01-Nx05 were 98.53 to 99.64% identical to the corresponding DNA sequences of Aphanomyces mitsuba based on GenBank BLASTn analysis and were deposited in GenBank (PQ219397-PQ219401, PQ228177-PQ228191). Phylogenetic analysis showed that all the five isolates were placed in A. mitsuba group, with bootstrap value of 100. Morphological, molecular and pathogenicity results confirmed that A. mitsuba can cause root rot of tobacco. A. mitsuba can cause stem and petiole rot of hydroponically grown Cryptotaenia japonica (Kageyama et al. 2023). To our knowledge, this is the first report of A. mitsuba infect of tobacco in field in China, showing that A. mitsuba had potential danger to crops in this country. This study enriched the information of tobacco pathogens and laid the foundation for further study of A. mitsuba.
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