First Report of Fusarium oxysporum Causing Vascular Wilt of Stevia rebaudiana in Colombia

Stevia (Stevia rebaudiana Bertoni) is a perennial-herbaceous plant with increasing global demand based on its powerful sweetening capacity free of calories. This Colombian crop achieves important commercial interest because of its large yield and sweet-tasting compounds (steviol glycosides) (Angelini et al. 2016; Pereira et al. 2016). In September 2017, diseased stevia plants were observed in a commercial plantation for leaf production located in Olaya, Antioquia, Colombia (6°35′18″ N, 75°46′55″ W). Symptoms including chlorosis and necrosis in leaves, stem rot, and global wilting occurred in plants after 6 weeks of vegetative growth stage. Fungal isolation was performed from both plants and soil. Four stems from decayed plants with vascular necrosis were surface sterilized and placed on potato dextrose agar (PDA) plates with oxytetracycline (0.9 ml/liter). Rhizosphere soil from four symptomatic plants was sampled, sieved, and then homogenized with sterile peptone/water 0.1% at 1:10 (w/v). Serial dilutions were plated on PDA/oxytetracycline plates. Both sets of plates (diseased plants and rhizosphere soil) were incubated for 3 to 5 days at 24 ± 2°C and 12-h light/12-h dark and were subjected to morphological analyses. For conidia formation, plates were incubated for 8 days at 28 ± 2°C in the dark, and recovered conidia were observed under optical microscope. Seven colonies of white aerial mycelia, slightly pinkish, were isolated into pure culture and were identified as Fusarium spp. Mycelium from pure cultures of isolates UDEAGIEM-H11 (rhizosphere soil) and CBS 145586 (diseased plant) were scraped with a sterile scalpel, and DNA was extracted. Polymerase chain reaction amplifications of ITS, EF, and β-tubulin regions were sequenced and exhibited 100% coverage and identity with that of Fusarium oxysporum after BLAST analysis. BLAST search of the ITS sequence (GenBank accession no. MK432913) showed 100% identity with F. oxysporum strain MF22472 (accession no. MH911412). EF and β-tubulin sequences (accession nos. MK432915 and MK432917, respectively) showed 100% identity with F. oxysporum isolate FPOST-36 (accession no. KX215033) and F. oxysporum f. sp. momordicae isolate GuangX33 (accession no. MF445645), respectively. Pathogenicity of isolate UDEAGIEM-H11 was tested on 2-month-old stevia seedlings. A conidial suspension (1 × 10⁶ conidia/ml) was prepared for root-dipping inoculation of stevia plants for 1 min. Plants inoculated with sterile distilled water served as controls. Ten plants were used per treatment (inoculated and control), which were placed in a greenhouse at 28 ± 2°C under moist conditions. Disease symptoms similar to those observed in the field appeared on 100% of the inoculated plants. Four to 6 days after inoculation, inoculated plants presented leaf chlorosis and necrosis and stem necrosis from roots to shoots that ended with the sudden death of seedlings 7 days postinoculation. Noninoculated plants remained symptomless under identical conditions. F. oxysporum was successfully reisolated from stems of symptomatic plants and identified by morphological characteristics and microscopic examination, fulfilling Koch’s postulates. To our knowledge, this is the first report of F. oxysporum causing Fusarium wilt of stevia in Colombia. The disease has previously been reported in Egypt, Venezuela, and Mexico (Hilal and Baiuomy 2000; Leyva-Mir et al. 2018; Salazar et al. 2015). The information that this disease has been detected in stevia cultures in Colombia is important owing to the risk of spreading symptoms of F. oxysporum in this important commercial crop. Management strategies should be implemented to reduce the potential economic losses in stevia producing areas of Colombia.