First Report of Pseudocercospora oenotherae Causing Leaf Spot in Rhinacanthus nasutus in China

Rhinacanthus nasutus (L.) Kurz is a traditional Chinese medicine in China. In August 2022, leaf spots were observed on R. nasutus in a Chinese herbal garden in Zhanjiang, Guangdong Province, China (21°17'30″N, 110°18'25″E). Disease incidence was 90% (n = 100 investigated plants from about 800 plants). The yellow spots were round, gray in the center and scattered on the leaves. The coalescence of the individual spot eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The tissue surface was disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-eight isolates were obtained (isolation frequency = 28/4 × 10 = 70%). Three representative single-spore isolates (RNPO-1, RNPO-2, and RNPO-3) by a single-spore isolation method (Fang. 1998) were used for further study. The colonies of isolates on PDA were olive green in 7 days at 28 °C. Conidiogenous cells were unbranched, geniculate-sinuous, tapered toward the apex, and 10 to 20 × 3 μm (n = 20). Conidia were solitary, smooth, straight or curved, pale brown, 3 to 8-septate, apex acute, base truncate, and 50.5 to 88.5 × 2.0 to 3.5 μm (n = 50). For molecular identification, the colony PCR method with Taq DNA polymerase and MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), actin (ACT) , and RNA polymerase II second largest subunit (RPB2) loci of the isolates using primer pairs ITS1/ITS4, EF1/EF2, ACT-512F/ACT-783R, and RPB2-7CF/fRPB2-11aR, respectively (O'Donnell et al. 1998; O'Donnell et al. 2010). Their sequences were deposited in GenBank under nos. OP963568 to OP963570 (ITS), OP998376 to OP998378 (TEF1), OP998373 to OP998375 (ACT), and OP998379 to OP998381 (RPB2). By using the Maximum Likelihood method, a phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, ACT, and RPB2 that clustered the isolates with P. oenotherae (the type strain CBS 131920). The fungus was thus identified as P. oenotherae basing on these morphological and molecular characteristics (Guo and Liu. 1992; Kirschner. 2015). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 28 °C to 30 °C. Healthy plants of R. nasutus were grown in pots, with one plant in each pot. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) of the isolates for about 15 s before they were adhered to the leaves for 3 days. Each isolate was inoculated with three plants (2 month old), and each plant was inoculated with five leaves. Sterile distilled water was as the control and same treatment. The test was performed three times. Symptom were found on the inoculated plants after 2 weeks with the disease incidence 100%, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. No pathogen was isolated from the control plants. P. oenotherae caused leaf spot on Oenothera biennis L. (Guo and Liu. 1992). To the best of our knowledge, this is the first report that R. nasutus is the new host of P. oenotherae (Crous et al. 2013). Thus, this work provides an important reference for the control of this disease in the future.