First Report of Fusarium oxysporum f. sp. cubense Race 4 Causing Fusarium Wilt Disease of Banana in Turkey

Cavendish banana is a valuable agricultural crop along the Mediterranean Coast of Turkey with 369,000 t of production, which meets 67.7% of domestic demand (Turkish Statistical Institute 2018). During a survey of banana greenhouses in Alanya, Anamur, and Gazipasa cities along the Mediterranean coast, plants showing severe wilt symptoms and collapse were detected in March 2018. Yellowing of the oldest leaves, which split at their base, brownish streaks of the vascular tissue of pseudostems, and root necrosis were observed on Cavendish ‘Grand Naine’. Disease incidence in the greenhouses in Alanya, Anamur, and Gazipasa cities was calculated as 17.8, 86.2, and 10%, respectively. Symptomatic tissues (140 pieces for each greenhouse) were surface sterilized with 2.5% NaOCl solution for 3 min, rinsed in sterile distilled water twice, and placed onto potato dextrose agar (PDA) with 150 mg/liter of streptomycin sulfate to isolate fungal pathogens. Fusarium-like colonies were obtained from the majority of tissues (72%), and single spores were subcultured onto fresh PDA for accurate identification. After 25 days of incubation (at 24°C in the dark), white, aerial, fast-growing colonies developed with a purplish bottom-surface color. Morphologically, these colonies resembled the published descriptions of Fusarium oxysporum Schlecht. emend. Snyd. & Hans. (Nelson et al. 1983) with their abundant microconidia generally single-celled, oval microconidia (average 7.9 × 2.4 µm, n = 50), borne in false heads, on simple short monophialides, presence of terminal or intercalary chlamydospores and thin-walled, septate (three to four celled) macroconidia (average 29.7 × 42.6 µm, n = 50). For molecular identification, DNA was extracted from mycelia and ribosomal RNA intergenic spacer regions were amplified with PCR using tropical race 4-specific primers (FocTR4-F/FocTR4-R) (Dita et al. 2010). The reference PCR products of F. oxysporum f. sp. cubense race 4 (obtained from the Jordan University, courtesy of Nida Salem) and F. oxysporum (nonpathogenic grapevine isolate) were used as positive and negative controls, respectively. After gel electrophoresis, the 462-bp amplicons were obtained from the isolates (BMAE70Foc, BMAE87Foc, BMAE104Foc, and the positive control) but not from the negative control. The sequenced PCR products were compared with the NCBI GenBank database. NCBI BLAST analysis showed 100% identity with the sequence of MG211816 (F. oxysporum f. sp. cubense strain VCG 01213) in the NCBI database. After phylogenetic analysis, they were found to be F. oxysporum f. sp. cubense race 4, and the sequences were deposited as accessions MN419031, MN419032, and MN419033. Pathogenicity tests were conducted under greenhouse conditions (27°C, 16 h/8 h day/night, 85% relative humidity) on healthy banana plants. Nine-week-old tissue-culture plantlets of Cavendish cultivar Grand Naine were inoculated by dipping their wounded roots for 30 min in a spore suspension (10⁶ conidia/ml) of the isolates (Dita et al. 2010). Control plants were similarly inoculated with sterile distilled water. Six plants for each of the four isolates, plus the controls, were placed in pots containing sand, peat moss, and soil, and they were grown in the greenhouse for 45 days. The inoculated plants revealed typical wilt and pseudostem necrosis, but no symptoms were observed from the plants inoculated with grapevine isolate of F. oxysporum and sterile water. Koch’s postulates were confirmed by reisolation (with 63.2% frequency) of the isolates from the inoculated plants and confirmed by molecular tests. To our knowledge, this is the first report of F. oxysporum f. sp. cubense race 4 in Turkey, and this report is an important finding to facilitate additional surveys to evaluate prevalence of this devastating disease of bananas in Turkey.