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First Report of Mature Citrus Trees Being Affected by Fusarium Wilt in Tunisia

Citrus is an important crop in Tunisia and over 98% of trees of all varieties are grafted on sour orange rootstock. Since September 2010, unusual wilt symptoms have been observed in Takilsa, Bni-Khaled, and Manzel Bouzalfa fields that eventually caused tree death. The disease was observed on 10- to 25-year-old trees of sweet orange (Citrus sinensis) ‘Washington Navel’ and on ‘Clementine’ tangerines (C. tangerina) ‘Cassar,’ ‘Hernandina,’ and ‘MA3,’ all grafted on sour orange (C. aurantum) ‘Bigarade Gou Tou.’ The most conspicuous symptoms were wilting of sections of the canopy, chlorosis and epinasty of young leaves, and discoloration of vascular tissue. No root rot was observed. The problem was widespread with a disease incidence of 45 to 67%. Similar symptoms were described by Timmer et al. (2) on Mexican lime (C. aurantiifolia) nursery plants and some other species of citrus. Three representative isolates of Fusarium oxysporum Schlechtend.:Fr. from crown were single-spored and identified by the production of characteristic, three- to five-celled, sickle-shaped macroconidia with foot-shaped basal cells, ellipsoid microconidia borne in false heads on short monophialides, and chlamydospores in culture (1). The internal transcribed spacer (ITS) region of rDNA and the elongation factor (TEF 1-α) were amplified with primers ITS1/ITS4 and (TEF1/TEF2), respectively. GenBank accessions of ITS region are KC282838, KC282839, and KC282840, for TEF 1-α region are KF531633, KF537336, and KF537337, showed 99% homology with isolates of F. oxysporum in Fusarium-ID data. Pathogenicity tests were conducted on 7-month-old seedlings of sour orange using 10 plants for each of the three isolates. Prior to inoculation, roots were scraping with a sterile scalpel and plants were dipped in a conidial suspension of F. oxysporum (10⁶ conidia ml^–1) for 10 min. Each seedling was planted in a separate pot containing 0.7 liter of sterile soil. Non-inoculated plants with scraped roots dipped in sterile distilled water served as controls. Plants were irrigated and placed in a greenhouse at 24 ± 2°C and 12-h photoperiod. One month after inoculation, leaf chlorosis was observed and 2 months later, 90% of inoculated plants presented a severe wilt. Symptoms on infected plants were similar to those observed in the field. F. oxysporum was successfully re-isolated from the stems, thereby completing Koch's postulates. Genomic DNA was isolated from the re-isolations and PCR amplification of the ITS region was performed with the same primers. There was 100% nucleotide identity with sequences of the original isolates. To our knowledge, this is the first report of fusarium wilt of citrus trees in Tunisia. The pathogen may represent a new form species because previously, the disease was only reported from lime and lemon.

References: (1) J. F. Leslie and B. A. Summerell. Page 256 in: The Fusarium Laboratory Manual. Blackwell Publishing Professional, Hoboken, NJ, 2006. (2) L. W. Timmer et al. Phytopathology 72:698, 1982.