LAUSR

Pachira glabra Pasq.is an important landscape tree in southern China due to its ornamental value. Between March and April - 2021, anthracnose-like symptoms on P. glabra leaves were found in the botanical garden (27.904°N, 112.918°E) of Hunan University of Science and Technology located in Xiangtan of Hunan Province. Over 700 plants were evaluated, and up to 30% of the plants were symptomatic. On each plant, approximately 22% leaves had symptoms. Disease severity was estimated to be 15.6 ± 6.1% (n=100) in moderately diseased plants. Initially, subcircular or irregular shaped, water-soaked spots with pale green to yellow centers appeared mostly along leaf margins. Later, theses spots turned light brown to dark brown with black borders, gradually enlarged, and often coalesced into large sunken, necrotic areas, leading to early leaf death and abscission. Thirty lesions (2 × 2 mm) collected from ten trees were sterilized in 75% ethanol for 10 s, 2% sodium hypochlorite for 30 s, rinsed in sterile water three times, placed on potato dextrose agar (PDA) with lactic acid (3 ml/liter), and incubated at 28°C for 5 days. After incubation, six isolates with a similar morphology were obtained by single-sporing. Colonies on PDA were white and with age produced a light brown pigmentation on the underside of the colony. Acervuli present in aged cultures, brown to black, circular to subcircular and measured 31.9 to 108.7 µm (71.4 ± 6.2 µm, n=30). Conidia were single-celled, transparent, smooth, fusiform to cylindrical with obtuse to slightly ronded ends, and measured 7.8 to 11.1 µm long and 2.5 to 3.1 µm wide (9.3 ± 1.0 × 2.9 ± 0.7, n=100). For further molecular identification, Internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate (GAPDH), calmodulin (CAL), and beta-tubulin (TUB2) genes of the isolates were amplified from genomic DNA, using primers ITS1/ITS4 (Mills et al. 1992), GDF/GDR (Cannon et al. 2012), ACT-512F/ACT-783R, CL1CF/CL2CR (Weir et al. 2012), and T1F/T22R (O'Donnell et al. 1997), respectively. Sequences of ITS (accession no. OM074029), ACT (OM190777), GAPDH (OM190778), CAL (ON210110), and TUB2 (ON210109) from CS-1 showed >98% identity where sequences overlapped to the reference strain of Colletotrichum siamense CBS 130420 (JX010259.1, JX009549.1, JX009974.1, JX009713.1 and JX010415.1). Concatenated sequences were used for a phylogenetic analysis based on Maximum Likelihood using MEGA-X. Based on morphological and molecular data, isolate CS-1 was identified as C. siamense (Cannon et al. 2012). . Pathogenicity tests were performed three times on healthy leaves using isolate CS1. Ten leaves on one-year-old plants were either slightly wounded by a sterile needle or unwounded, and inoculated with 10 µl of conidial suspension (1×106 conidia/ml, containing 0.05% Tween 20) per wound. The control plants were treated with sterile water. All plants were kept in a greenhouse for 24 h at 28°C and 80% relative humidity, with a 12-h photoperiod and then transferred to natural conditions. All wounded, inoculated leaves developed leaf spot symptoms after 14 days similar to those observed in the field, whereas no visible symptoms appeared on the intact and noninoculated leaves. C. siamense strains were reisolated from all symptomatic leaves, fulfilling Koch's postulates. C. siamense has been reported as a causal agent of anthracnose associated with diverse species (Udayanga et al. 2013), but not including P. glabra. To our knowledge, this is the first report of C. siamense causing anthracnose on P. glabra.