First Report of Charcoal Rot of Stevia Caused by Macrophomina phaseolina in North Carolina

Stevia (Stevia rebaudiana [Bertoni] Bertoni) is an emerging perennial crop in the United States. Small black root lesions were first observed on overwintering crowns dug in March 2016 from a field planting of stevia in Rocky Mount, NC. Similar root lesions were observed on first year plants in Kinston and Rocky Mount, NC, throughout the 2016 growing season in fields with a history of soybean and corn rotations. Isolations from lesions were made on potato dextrose agar amended with 50 µg/ml of streptomycin sulfate and penicillin G. Isolations were incubated at room temperature (22 to 25°C) for 1 week and observed for colony morphology. Gray-black colonies that became dark with age were isolated into pure culture. Hyphae were septate with barrel-shaped cells, and numerous 50 to 150 µm microsclerotia resembling those produced by Macrophomina (Kaur et al. 2012) were present. Pathogen identification was confirmed by sequencing the internal spacer (ITS) region of ribosomal DNA. DNA was extracted from mycelia with the Qiagen DNEasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to a PCR using universal primers ITS 4,5. Isolates were identified as Macrophomina phaseolina (100% sequence identity with GenBank accession no. KF951775.1). Koch’s postulates were confirmed on 10-week-old stevia plants, cv. G3, grown in 8-cm-diameter pots in the greenhouse. Ten 6.75-mm agar plugs were buried 1 cm deep approximately 2 cm from the base of the plant in each of six replicate sterile pots. Plants were observed over a 6-week period for symptom development, at which time root necrosis similar to field symptoms was evident on all inoculated plants. Noninoculated plants did not develop symptoms. M. phaseolina was reisolated from necrotic inoculated roots and confirmed by morphology and sequencing. To our knowledge, this is the first report of M. phaseolina causing charcoal rot of stevia in North America. Hilal and Baiuomy (2000) reported M. phaseolina as the causal agent of charcoal rot on stevia in Egypt. At this time, no fungicides are registered for stevia. Since stevia is often grown as a perennial rotation crop, and M. phaseolina was present year-round in roots of stevia, it will be important to determine if this pathogen becomes more damaging over a three year production cycle and reaches levels that may have negative impacts on rotation crops.