First Report of Leaf Spot Caused by Colletotrichum siamense on Pharbitis purpurea in Sichuan, China

The Pharbitis purpurea (L.) Voisgt, a member of the Convolvulaceae, is a graceful plant with an air purifying function and ornamental values. It is often cultivated in parks and roadsides. In April 2021, leaf spots (with approximately 67.9% disease incidence) were observed on P. purpurea grown in Xichang city (27°49'N; 102°16'E). More than 1000 square meters of planting area were investigated. Initially, yellowish-brown spots were of different sizes with a yellow irregular border, and slightly sunken necrotic lesions. Gradually, the necrotic lesions expanded and developed into brown spots that often coalesced and expanded to cover the entire leaves. Finally, the leaves wilted, died and fell off. For fungal isolation, infected tissues from ten samples were cut into small pieces of (2.5 × 2.5 mm) sterilized with 3% NaOCl for 30 s and 75% ethanol for 60 s, rinsed three times with sterilized water, blot-dried and cultured on potato dextrose agar (PDA) at 25°C in dark for 8 days. After culturing for 8 days, the colony diameter reached 75.2 to 79.7 mm. The pure colonies were grayish-white with pale yellowish borders and grayish black and pale yellowish borders on the reverse side. The conidia were hyaline, single-celled, cylindrical, smooth-walled, subcylindrical with obtuse to slightly rounded ends, measuring 11.6 to 17.9 × 3.7 to 5.8 μm (n = 100; average=14.7 × 4.9μm). These morphological characteristics were consistent with the description of Colletotricum siamense (Zhang et al. 2021). For molecular identification, the genomic DNA of the representative isolate LBH202104 was extracted using a fungal genomic DNA extraction kit (Solarbio, Beijing). Partial of internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, and GDF/GDR, respectively (Weir et al. 2012). BLAST results of obtained sequences (ITS: OM948680, ACT: OM959361, CAL: OM959366, and GAPDH: OM959364), showed99% identity with C. siamense sequences (MN305712, MZ461478, MK141754, and MK361203) in GenBank. Based on morphology and phylogenetic analysis, the representative isolate was identified as Colletotrichum siamense (Fig. S1S2). For pathogenicity test, the conidial suspension (1 × 106 conidia/ml) was sprayed on the leaves of 4-year-old eight potted P. purpurea plants. Fifteen leaves of each plant were inoculated. For negative controls, 8 plants were sprayed with sterilized distilled water. Finally, all pots were kept in a greenhouse at 26°C under a 16 h/8 h photoperiod and 68 to 75% relative humidity. The inoculated plants showed symptoms similar to those of the original diseased plants, while controls remained asymptomatic. C. siamense cultures were re-isolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated thrice, which showed similar results, confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. siamense on P. purpurea worldwide. The identification of this pathogen provides a foundation for the management of Leaf spot in P. purpurea.