Bixa orellana L. is a traditional Chinese medicine. In December 2019, a leaf spot disease was observed on B. orellana from a field in Zhanjiang (21°18'12''N, 110°17'22''E), China. Disease incidence was around 85% (n = 100 investigated plants from about 30 hectares). Initial leaf spots were circular, and the center of the lesions was grayish-white with a purple black border. The coalescence of individual spots 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, and the surfaces were disinfected with 75% ethanol for 30 sec, and 2% sodium hypochlorite for 60 sec. The the samples were then rinsed three times in sterile water, plated on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Three representative isolates (BOPP-1, BOPP-2, and BOPP-3) were used for further study. The colonies of isolates on PDA were dark olive green with off-white aerial mycelia after 7 days at 28 °C. Conidia were solitary, smooth to verrucous, olive to light brown, slightly curved, narrowly obclavate, apex obtuse, base obconic-truncate, had 2-4 septa, and 30.4-55.5×2.0-3.5 μm in size.. These morphological characteristics showed did not differ from the description of Pseudocercospora paraguayensis (Crous et al. 1997). For molecular identification, the internal transcribed spacer (ITS) region, the translation elongation factor 1-α (TEF1) gene, and actin (ACT) gene were amplified using primer pairs ITS1/ITS4 (White et al., 1990), EF1/EF2 ( O'Donnell et al. 1998), and ACT-512F/ACT-783R (Carbone and Kohn, 1999) and sequenced from DNA extracted from the three isolates, respectively. Sequences were deposited in GenBank under accession no. MZ363823-MZ363825 (ITS), MZ614954-MZ614956 (TEF1), and MZ614951-MZ614953 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that the three isolates were nested within the clade containing the type specimen of P. paraguayensis (CBS 111286) but not within P. bixae (the type specimen CPC 25244). Pathogenicity was tested through in vivo experiments. Inoculation and control seedlings (n = 5, 1-month-old) were sprayed with a spore suspension (1 × 105 per ml) of P. paraguayensis and sterile distilled water (control), respectively, until run-off (Fang. 1998). The plants were grown in pots in a greenhouse at 28°C, with at approximately 80% RH. The test was performed three times. Symptoms similar to those in the field were observed on the inoculated plants after two weeks. The control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and comparison of ITS sequences with 100% identical to those of isolates. No original fungi were isolated from the control plants. A previous study reported that P. paraguayensis caused leaf spots on pistachio and eucalypts, and the fungus causing the leaf spots of B. orellana was redescribed as P. bixae (Crous et al. 2019). However, multilocus phylogenetic analyses differentiated P. paraguayensis from P. bixae. In the present study, P. paraguayensis was distinguished from P. bixae due to the absence of catenulate conidia and the presence of finely verruculose conidia (Crous et al. 2013). P. eucalypti as a synonyms was reported in Taiwan (www.MycoBank.org). The current study is the first to report P. paraguayensis causing leaf spots on B. orellana from Chinese Mainland. This finding will help to provide a scientific basis for the disease detection.
Keywords: Bixa orellana; Leaf Spots; Pseudocercospora paraguayensis.