First Report of Leaf Spot Caused by Colletotrichum siamense on Philodendron bipinnatifidum in Guangxi, China

Abstract

Philodendron bipinnatifidum belongs to the Araceae family, as its graceful plant architecture and the function of purifying air and water, which has important ornamental value (Yu et al. 2019). P. bipinnatifidum is often cultivated in parks and along roadsides. In August 2019 and June 2020, leaf spot disease was observed on over 80% of P. bipinnatifidum plants in Qingxiushan Park (N22°47′23.35″, E108°23′4.26″), Nanning, Guangxi, China. The disease was also frequently observed on P. bipinnatifidum in some other places in Nanning. Symptoms began as small, round, brown spots. As the disease developed, the center of the lesions was sunken with a dark brown border (Fig. 1). Under severe conditions, some spots were joined into larger irregular spots, and even whole leaves died. For fungal isolation, small pieces (5 × 5 mm) were cut from the margin between lesion and healthy tissues of symptomatic leaves, disinfected with 75% ethanol for 10 s, 1% sodium hypochlorite solution for 1 min and washed by sterile water for three times. Over one hundred morphologically similar colonies with white mycelia and a dark green pigment were obtained after 5 days incubation on potato dextrose agar (PDA) at 25°C. Isolates GBZ6-1, GBZ9-1 and GBZ9-2 were selected for intensive study, and the mycelial growth rates of them averaged 14.5 mm/day, 14.6 mm/day and 13.2 mm/day, respectively. Isolates could produce orange conidia on PDA for 7 days. Conidia were elliptical, aseptate and colourless, with sizes of 15.4 ± 0.14 µm × 5.8 ± 0.1 µm, 15.6 ± 0.14 µm × 5.3 ± 0.1 µm and 15.0 ± 0.16 µm × 6.3 ± 0.1 µm for GBZ6-1, GBZ9-1 and GBZ9-2, respectively. Appressoria were mostly brown ovoid, conidial appressoria averaged 7.62-9.31 μm × 5.84-742 μm, and mycelial appressoria were 8.06-9.22 μm × 6.28-6.92 μm. Genomic DNA was extracted by the DNAsecure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd] and the rDNA internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes (Weir et al. 2012) were sequenced. Phylogenetic analysis was performed using RAXML (Version 2.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, and GAPDH). The results showed that three isolates were identified as C. siamense (accessions MW116805-MW116816, for ACT, CAL, CHS-1 and GAPDH of GBZ6-1, GBZ9-1 and GBZ9-2; MW073391-MW073393 for ITS of GBZ6-1, GBZ9-1 and GBZ9-2). According to the cultural and conidial morphology, as well as phylogenetic analysis, these isolates were identified as Colletotrichum siamense. Pathogenicity tests were conducted on one-year-old plants. Young healthy leaves of three plants were artificially wounded by gently scratching with a sterile needle and 10 µl of conidial suspension (106 spores/ml) were applied to per wound site for each isolate. Wounded leaves were inoculated with 10 µl of water as controls. All plants were sprayed with water and covered with plastic bags to maintain high humidity. Symptomatic lesions were observed on the inoculated leaves after 15 days at 28 °C, whereas no symptoms were observed on the control leaves. To fulfill Koch’s postulates, fungi were reisolated from symptomatic leaves and morphologically identical to the inoculated isolates (Xue et al. 2020). To our knowledge, this is the first report of leaf spot caused by C. siamense on P. bipinnatifidum worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. siamense on P. bipinnatifidum.