First Report of Sclerotinia Blight Caused by Sclerotinia sclerotiorum on Oriental Tobacco in the Yunnan Province of China

Abstract

Yunnan Province is a major oriental tobacco (OT) production area with >90% of China’s output (Qu et al. 2014). In the spring of 2018 and 2019, we conducted OT disease surveys in Mengtong, Baoshan, Yunnan Province, and we found an unknown disease displaying rotting, wilting, and blighting in two fields (24°32′37″ N, 99°37′34″ E; 24°32′46″ N, 99°37′31″ E; ∼2,000 m²). Average incidence was 3.0 and 8.4%, respectively. Disease development began with water-soaked, oval or circular spots with white centers and olive-green edges that formed on the wounds of stems and petioles near the soil, and infected stems subsequently became bleached and died. In the late phases, white cottony mycelium appeared on the dead tissues. Spherical to cylindrical black sclerotia (5.0 to 17.0 × 3 to 7 mm; avg. 9.1 × 4.5 mm; n = 50) were observed inside and on the surface of stems. To isolate the pathogen, five and four infected samples were collected from fields in 2018 and 2019, respectively. Tissue sections (5 mm²) with typical symptoms were surface disinfected in 75% ethanol for 5 s and 0.5% NaOCl for 1.5 min, rinsed in sterilized distilled water three times, and dried. Tissues were placed on PDA plates and cultured at 25°C for 3 days in the dark. Hyphal tips were transferred to fresh PDA plates. Thin, cottony-white, fluffy mycelia grew to cover the Petri dish (9 cm diam.) in 3 to 4 days. Irregular white immature sclerotia were produced 4 to 5 days later on the edges of the plates. After 7 to 14 days, abundant spherical or oval black sclerotia of 2.4 to 5.0 × 1.4 to 3.8 mm (avg. 3.4 × 2.6 mm, n = 50) were formed. Morphology of all isolates was similar to Sclerotinia sclerotiorum (Lib.) de Bary. To confirm the pathogen, we extracted genomic DNA from the mycelia of isolate HP-1 using a Fungal DNA Extraction Kit (Omega). The ITS region and partial β-tubulin gene sequence of HP-1 were amplified with primers ITS1/ITS4 and Bt2a/Bt2b, respectively (Doyle and Doyle 1987; Glass and Donaldson 1995). PCR amplicons were purified and cloned into pMD19-T simple vector (Takara Biomedical) for Sanger dideoxy sequencing at Shanghai Life Technology Co. BLASTn search revealed the 462-bp ITS sequence (MK656936.1) was identical to S. sclerotiorum (MK527226.1, MK010971.1, MG931017.1, etc.), and the 472-bp partial β-tubulin sequence (MN295984.1) was too (MG931018.1, KX781302.1, KF545201.1, etc.). Koch’s postulates were fulfilled by inoculation and reisolation of HP-1 from five plants. Five mycelial plugs, 8 mm diameter, from 5-day-old culture grown on PDA were used to inoculate the lower stems of five 45-day-old susceptible plants (BASMA-1) and wrapped with wet sterilized cotton. PDA plugs without fungi were controls. Plants were maintained in a greenhouse at 25°C with a 12-h photoperiod. Five days after inoculation, all inoculated stems became water-soaked brown and covered with white mycelia. Symptoms typical of Sclerotinia blight (SB) on stems were observed 10 days after inoculation. S. sclerotiorum was consistently reisolated. No symptoms developed on the controls. Similar results were obtained in three independent experiments. Seedling blight caused by S. sclerotiorum has been recorded on flue-cured tobacco in Brazil, the United States, and Jilin and Anhui Provinces of China (Gao et al. 2018; Farr and Rossman 2019; Qian et al. 1994). This is the first observation of SB on OT in Yunnan Province. OT (Nicotiana tabacum L.) is a sun-cured, highly aromatic, small-leafed variety planted under dry and hot conditions from November to April in Baoshan, where rainfall increases from February to April. SB is a major disease of OT in this area; however, it has not been reported on flue-cured tobacco in Yunnan Province. SB of OT is often misidentified as black shank caused by Phytophthora nicotianae, the most common disease on flue-cured tobacco in Yunnan Province (Zhang and Chen 1999). This report will aid local farmers and tobacco technicians in accurate diagnosis and control of the disease with more appropriate measures.