First Report of Colletotrichum siamense Causing Leaf Anthracnose on Cotton in India (original) (raw)

Rosa chinensis Jacq. is a traditional Chinese ornamental plant extensively cultivated in China. In April 2018, anthracnose lesions were found on leaves of R. chinensis planted in a garden in Changsha City, China. The symptoms initially appeared as small circular, sunken necrotic lesions. The center of lesions were gray with a pale brown border. As the disease progressed, lesions expanded into 5 to 14-mm-diameter spots and were near round or irregular. To identify the pathogen, pieces of symptomatic tissues were surface-sterilized for 1 min in 1% NaClO, plated on potato-dextrose agar (PDA) with 0.1 mg/ml of ampicillin, and incubated at 25 ± 2℃ in the dark. A Colletotrichum sp. was consistently isolated from diseased tissues. Three different single-spore cultures (RS29, RS30, RS35) were used for fungal morphological characterization and identification. The three isolates showed the same morphological features: colony mycelium was initially white, turning pale gray with time, while the reverse side was dark brown at the center of the plate (Fig 1A-B). The average growth of colonies was 9.8 mm per day (n=7). The hyphae were hyaline. Conidia were straight, hyaline, fusiform to cylindrical, with rounded apices, and ranged from 11.4 to 17.8 μm (mean 14.5 μm) × 2.7 to 5.5 μm (mean 3.8 μm) (n=100) (Fig 1C-D). Appressoria were single or clustered, variable in shape, brown, smooth-walled, and ranged from 4.7 to 6.6 μm (mean 5.5 μm) × 3.6 to 5.4 μm (mean 4.4 μm) (n=50) (Fig 1E). The morphological and cultural traits were consistent with descriptions of C. siamense (Prihastuti et al. 2009; Sharma et al. 2013). To confirm identification, partial sequences of the internal transcribed spacer (ITS), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), glutamine synthase (GS), and β-tubulin 2 (beta-TUB-2) genes of the three isolates were obtained and deposited into GenBank with accession nos. MH643994-MH643999 and MK446249-MK446260. A phylogenetic tree generated by neighbor joining using the six gene sequences in MEGA 7 revealed that our isolates clustered in the same clade with C. siamense (Fig. 2) (Weir et al. 2012). Mycelial plugs (5 mm in diameter) from a 7-day-old colony of RS29 were inoculated on ten wounded (using sterile needle) and ten non-wounded leaves, stems, receptacles and calyxes of healthy R. chinensis plants that were previously disinfested with 1% NaClO and rinsed twice with sterile distilled water, respectively. An equal number of plants inoculated with plugs of non-colonized PDA served as the control. Pathogenicity tests were repeated three times. Plants were kept under greenhouse conditions at 25 ± 2℃ and 90% relative humidity on a 12-h fluorescent light/dark regimen. Initial symptoms resembling those in the garden appeared after 3 days on all wounded and inoculated plant tissues, and after 9 days on non-wounded inoculated plant tissues, whereas the control plants remained healthy (Fig 1F-I). The pathogen was re-isolated from inoculated plants, completing Koch's postulates. This is the first report of C. siamense in China causing anthracnose on R. chinensis. The identification of this pathogen provides a foundation for the management of anthracnose in R. chinensis.