First Report of Leaf Spot on <i>Pseudostellaria heterophylla</i> Caused by <i>Pestalotiopsis kenyama</i> in Guizhou, China

<i>Pseudostellaria heterophylla</i> is a perennial herb of Caryophylaceae family, cultivated in Guizhou and Jiangsu province of China. This herb is particularly important in Chinese herbal medicine, especially valued for its reputed benefits to the immune system (Choi et al. 2016). In May 2023, the incidence of <i>P. heterophylla</i> leaf spot was approximately 50% in Cengong County (108° 37' E, 27° 36' N), Guizhou Province, southwestern region of China. The symptoms gradually developed from small white lesions to larger lesions. Fifteen symptomatic leaves from different plants were collected and fungal isolates were obtained by a single spore method (Liu 2010). Briefly, the conidia on the leaves were collected in a sterile tube to prepare a conidial suspension, which was gradually diluted to a single spore under a microscope and then transferred to PDA at 28°C. Finally, a total of 18 isolates were obtained. Colonies reached 89 mm in diameter after 7 days of incubation at 28°C on PDA. It was white, irregular and had gregarious conidiomata measuring 100 - 400 µm. These conidia were fusiform, slightly curved, measuring 24.5 - 35.5 × 4.0 - 5.5 µm (n=50). The basal and apical cells are hyaline, with 1, 1-3 accessory filaments, respectively, and the middle three cells are light brown. The conidiogenous cell was discrete and nearly cylindrical. Based on the morphological characteristics, the isolates were initially identified as <i>Pestalotiopsis</i> (Liu et al. 2017). To confirm the identification, the internal transcribed spacer region (ITS) gene, large subunit (<i>LSU</i>), the translation elongation factor-1 (<i>TEF1</i>) gene, and the β-tubulin (<i>TUB2</i>) gene were amplified using ITS4/ITS5, LR5/LROR, EF1-728F/EF2, and Bt2a/T1 primers, respectively (Liu et al. 2017; Carbone et al. 1999; O' Donnell et al. 1998; Santos et al. 2010). The ITS, <i>LSU</i>, <i>TEF1</i> and <i>TUB2</i> sequences of three randomly selected isolates, GUCC0218 (OR782588, OR782589, OR791708 and OR791719), GUCC0220 (PP837725, PP837723, PP855992 and PP855993) and GUCC0221 (OR782591, OR791002, OR791709 and OR791720), showed 100% nucleotide identities with the sequences of <i>P. kenyama</i> (CBS 442.67) in GenBank (KM199302, KM116234, KM199502 and KM199395, respectively). The phylogenetic tree based on ITS, <i>LSU</i>, <i>TEF1</i> and <i>TUB2</i> sequences, using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (Bl) methods, indicated that three isolates were most closely related to <i>P. kenyama</i> (CBS 442.67), supported by 100%/1/99% bootstrap values. Nine 4-month-old healthy <i>P. heterophylla</i> plants were sprayed with a conidial suspension (1 × 106 conidia/mL) the three isolates and three other plants were sprayed with sterile water as a control, and all treated plants were incubated in a greenhouse at 28°C (85% humidity). After 7 days, typical symptoms similar to those observed in the field appeared on the leaves of inoculated plants, while the control plants remained asymptomatic. <i>Pestalotiopsis kenyama</i> was re-isolated from infected leaves but not from controls. <i>Pestalotiopsis kenyama</i> is accountable for <i>Camellia sinensis</i> gray blight (Liu et al. 2017), <i>Castanea mollissima</i> leaf spot (Jiang 2021). Our findings will provide a basis for the correct diagnosis and management of leaf spot on <i>P. heterophylla</i>. To our knowledge, this is the first report of leaf spot disease caused by <i>P. kenyama</i> on <i>P. heterophylla</i> in China.