First Report of Leaf Spot on Stephania tetrandra Caused by Albifimbria verrucaria in China.

Stephania tetrandra S. Moore belongs to the family Menispermaceae and is a Chinese medicinal plant widely distributed in tropical and subtropical regions of Asia and Africa. The root can be used for a variety of treatments (Jiang et al. 2020). In August 2021, leaf spot symptoms were observed on S. tetrandra cultivated in Jiangxi (114.456E, 27.379N, southern China). The disease symptoms included a slight constriction of the leaves, with irregularly shaped brown to black spots with well-defined borders. Severely affected leaves were shed by the plant. In order to determine the cause, symptomatic leaves were surface-disinfested with 0.6% NaOCl for 2 min, and rinsed twice in sterile water, then incubated on moist paper towels at 26°C in the dark for 2 days. Cream-colored sporodochia were observed within the leaf spots, turning dark green to black within 16 hours. A slow-growing white fungus was isolated from 95% of the samples (n = 30) on PDA. Dark green sporodochia emerged after 7 to 10 days of incubation, and released tip-end oval, non-septate, hyaline conidia measuring 6.7 to 8.5 μm (mean 7.5 μm, n = 50) by 2.0 to 3.3 μm (mean 2.7 μm, n = 50). Concentric rings were interspersed with sporodochia on the continually incubated mycelium. The morphological characteristics of the isolates matched the description of Albifimbria (Lombard et al. 2016). Nucleotide sequences, amplified from isolate FJL5C using primers of the internal transcribed spacer (ITS) (White et al. 1990), calmodulin (cmdA; Carbone and Kohn 1999), and RNA polymerase II second largest subunit (rpb2; O'Donnell et al. 2007), were deposited in GenBank under accession numbers OM317911, OM386815, and OM386816. A BLASTn analysis of the sequences showed 100% identity with the type strain CBS 328.52 (Lombard et al. 2016) of Albifimbria verrucaria (syn. Myrothecium verrucaria) for ITS, and 99% for cmdA and rpb2 (KU845893, KU845875, and KU845931, respectively). A phylogenetic tree generated using the three sequences showed that the isolate from S. tetrandra grouped with the A. verrucaria isolates, but away from other species of Albifimbria. These results together with the lack of a pale luteus exudate produced by A. viridis (Lombard et al. 2016) implied that the isolate was A. verrucaria. The culture was deposited in Guangdong Microbial Culture Collection Center (GDMCC 3.716). To verify pathogenicity, conidial suspension (106 conidia/mL in 0.05% Tween 20 solution) was sprayed onto six healthy plants. Six other plants sprayed with the Tween 20 solution alone served as controls. All plants were incubated in the dark at 26°C and 95% humidity for 30 hours, then transferred to a greenhouse at 26°C and 12 hours of illumination per day for 2 to 3 days. Inoculated leaves developed similar symptoms to those described above, whereas control plants remained healthy. The same pathogen was isolated from the diseased leaves, with the same morphological and molecular traits as those from the field plants. This experiment fulfilled Koch's postulates and confirmed that A. verrucaria causes leaf spots on S. tetrandra. This pathogen has been reported to cause disease in a wide range of weeds, legumes, and crop plants (Herman et al. 2020). To our knowledge, this is the first report of A. verrucaria causing leaf spots on S. tetrandra in natural or controlled environments. The disease can seriously threaten S. tetrandra on growth and yield loss.