First Report of Colletotrichum karstii Causing Anthracnose on Cigar Tobacco in Hainan, China

Abstract

Cigar tobacco (Nicotiana tabacum L.) is an important cash crop in China. Due to suitable ecological conditions, Hainan province is a major area for growing cigar tobacco. Cigar tobacco leaves are both an important source of fiscal taxation and also the main source of income for farmers in Hainan province. In 2018 and 2019, necrotic leaf spots were observed on 5-15% of cigar tobacco plants in Danzhou city (109.58°E, 19.53°N) and Wuzhishan city (109.52°E, 18.78°N) in Hainan province, China (eXtra Fig. 1). Lesions on plants in the fields were small, nearly round spots (0.2-0.5 cm in diameter) with light gray centers and narrow dark brown margins, and sometimes there were tiny black dots composed of sporophores scattering in the lesion center. To determine the causal agent, more than ten symptomatic leaves from Danzhou city and Wuzhishan city were used for isolation. Small pieces of tissue were cut from lesion borders, sterilized with 2% sodium hypochlorite (NaClO) for 4 mins and 70% ethanol for 30 s, rinsed with sterilized water (SW), and dried on a sterile tissue paper. Clean and excised tissue pieces were placed on potato dextrose agar (PDA) plates and incubated at 28°C for 5 days in the dark. Pure colonies of the fungus had grayish white aerial mycelia and orange conidial masses in the centers, and sometimes there were tiny black dots composed of clusters of conidiophores and conidia scattered in the centers. A pure culture was deposited in the China General Microbiological Culture Collection Center (CGMCC, No. 3.19502). Microscopic observations showed single, straight to slightly curved,/span>hyaline, cylindrical conidia that were 12.1-14.7 μm long and 6.4-8.2 μm wide (eXtra Fig. 2). To confirm the identities of the isolate, the internal transcribed spacer (ITS) rDNA of the fungus was amplified using PCR and sequenced, as described by Yang et al. (2011). The sequencing results were then compared with other DNA sequences in the GenBank database by BLAST. Analysis of the PCR product (GenBank Accession No. MK050853) showed that the sequence had 99.5% identity with the nucleotide sequences of Colletotrichum karstii (HM585409). Glyceraldehyde-3-phosphate dehydrogenase (GPDH, GDF1/GDR1) (Yang et al. 2011) was also sequenced and BLAST. The PCR product sequence had 99.8% identity with the nucleotide sequences of C. karstii (HM585391). Based on the morphological and molecular characteristics, the isolate CGMCC 3.19502 was identified as C. karstii. Pathogenicity tests were conducted by artificially inoculating twenty 40-day old cigar tobacco plants (cv. H211). A spore suspension (106 conidia/ml) was evenly sprayed on all leaves of the twenty plants. Ten control plants were treated similarly with SW. Twenty inoculated plants and ten non-inoculated plants were placed in a chamber maintained at 30°C and 90% RH. After 7 days, the same symptoms observed from natural infections were on inoculated leaves, while the controls remained symptomless. This isolate also infected detached tobacco leaves (eXtra Fig.3). The pathogen was re-isolated from inoculated leaves, and Koch’s postulates were completed by confirming the identity of the isolated fungus based on the morphology and ITS sequence. Anthracnose caused by C. karstii was previously detected on Schefflera octophylla in Sichuan province (Li et al. 2017). But to our knowledge, this is the first report of C. karstii causing anthracnose on cigar tobacco in China. Damage caused by C. karstii could cause significant economic losses if not managed. More research is needed to better understand this disease and establish control strategies.