LAUSR

Philodendron bipinnatifidum Schott ex Endl (Araceae) is native to South America. It was introduced in Guangdong around the 1980s, and then gradually promoted for use as a landscape ornamental in South China (You et al. 2013). Previous studies showed that an extract of P. bipinnatifidum displayed antinociceptive and anti-inflammatory activities (Scapinello et al. 2019). In August 2019 and June 2020, leaf spot disease was observed on P. bipinnatifidum leaves in Qingxiushan Park, Nanning, Guangxi province, China, with approximately 80% disease incidence. Symptoms began as small brown spots that extended into large, irregular, dark brown, necrotic, sunken lesions. The leaves eventually became yellow and then withered and died. The symptomatic leaves were sampled from three different places in the park. Leaf pieces (5× 5 mm) of three samples were cut from the junction of diseased and healthy leaf tissue, disinfected in 75% (v/v) alcohol for 10 sec, 2% (v/v) sodium hypochlorite for 1 min, and then rinsed three times in sterile distilled water before pieces were incubated on potato dextrose agar (PDA) at 25°C for 7 days. Eighty-one Colletotrichum isolates were obtained, with an 88% isolation rate, and three of these (GBZ7-1, GBZ7-3 and GBZ8-2) were selected for intensive study. After 7 days, the colonies on PDA showed white-to-gray aerial mycelium. Conidia (n=90) were elliptical, single-celled, hyaline, straight, 14.61 ± 0.08 µm × 6.84 ± 0.04 µm (C. karsti), and 15.15 ± 0.11 µm ×5.04 ± 0.04 µm (C. endophytica). Appressoria (n=90) were melanized, subglobose, irregular, 9.57 ± 0.17 µm × 7.18 ± 0.10 µm (C. karsti), and 7.36 ± 0.18 µm × 5.52 ± 0.13 µm (C. endophytica). To confirm morphological identification, the rDNA internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), β-tubulin 2 (TUB2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012) were amplified and sequenced (GenBank accessions Nos. ITS (MZ962373 ~ MZ962375), ACT (OK040200 ~ OK040202), CAL (OK040205 ~ OK040207), CHS-1 (OK040210 ~ OK040212), TUB2 (OK040220 ~ OK040222) and GAPDH (OK040215 ~ OK040217) of GBZ7-1, GBZ7-3 and GBZ8-2 respectively). Phylogenetic analysis was done using RAXML (Version 2.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, TUB2 and GAPDH). Isolates GBZ7-1 and GBZ7-3 were identified as C. karsti and GBZ8-2 as C. endophytica. Pathogenicity tests were performed with the three isolates on 45 asymptomatic attached leaves of nine one-year-old plants (three plants per isolate). Every leaf was punctured at three points using a sterile needle and inoculated with 10 µl of conidial suspension (106spores/ml) on each wound. Wounded leaves treated with sterilized water under the same conditions served as controls. The experiment was repeated three times. All plants were sprayed with water and covered with plastic bags to maintain high humidity. Sunken necrotic lesions were observed on all inoculated leaves after 15 days at 28 °C, whereas no symptoms were observed on the control leaves. C. karsti and C. endophytica were consistently re-isolated from the inoculated leaves which was confirmed by morphology and sequencing, fulfilling Koch's postulates. C. siamense was previously reported as a pathogen on P. bipinnatifidum in China (Ning et al. 2021). To our knowledge, this is the first report of leaf spot caused by C. karsti and C. endophytica on P. bipinnatifidum worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. karsti and C. endophytica on P. bipinnatifidum.