First report of Thielaviopsis punctulata causing black scorch disease on date palm in Qatar

Abstract

Ceratocystis radicicola (anamorph: Thielaviopsis paradoxa) was reported as an economically important pathogen causing serious diseases on date palm such as rhizosis (2) and black scorch (3) or as an associated pathogen with diseased date palm (1). In this study, we report for the first time that C. radicicola also causes black scorch disease in Qatar. In April to May 2013, we conducted a disease survey in 11 farms located in northern and southern Qatar where three infected farms had an average of 10% disease incidence. Infected trees manifested different disease symptoms such as black scorch of leaves, inflorescence blight, and heart and bud rot. Infected tissues were surface sterilized with 1.0% NaOCl for 60 s, rinsed with distilled water, blotted dry, and then plated on potato dextrose agar (PDA) supplemented with 50 mg/liter Rose Bengal. Single fungal colonies were picked from hyphal tips and grown on PDA for 7 days at 25°C for further examination of the mycological characteristics. Colonies of five C. radicicola isolates on PDA developed aerial mycelium with a light gray color in culture plate, which later changed to black. Both light and scanning electron microscopy were employed to delineate species by spore morphology. Colonies produced ovate aleuroconidia (14 to 17 × 9 to 12 μm) and cylindrical phialoconidia (7 to 9 × 3 to 4 μm) characteristic features of C. radicicola. Phialoconidia (endoconidia) were hyaline to brown in chains produced from endoconidiophore, clamydospores (aleuroconidia), which were single with smooth or slightly rough wall. Additionally, C. radicicola produced single alueroconidia from conidiophores. Amplification of ITS rDNA region from fungal genomic DNA of five isolates, using universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-CCTCCGCTTATTGATATGC-3′) confirmed the isolated fungus as C. radicicola with no intra-specific variation among the fungal isolates. The length of ITS-rDNA sequence was 534 bp (KJ410228) and had 99 and 93% sequence identity with ITS-rDNA region from C. radicicola (HQ443203) and C. paradoxa (HC415073.1), respectively. A pathogenicity test was conducted using 3-year-old trees from three cultivars (Khalas, Khneezi, and Barhi) growing in sandy loam soil under greenhouse conditions (25 to 29°C and 12/12-h light/dark). Six trees from each cultivar were used for pathogenicity test, where three were inoculated and three other mock-inoculated. Eight millimeter diameter mycelial plugs were obtained from a C. radicicola culture on PDA medium and used to inoculate rachis region and basal petioles of date palm leaves with a 9-mm wound created with a cork borer. Control plants were mock-inoculated with PDA plugs. The inoculated area was covered with wet cotton to prevent dryness and the whole plant was covered for 72 h. Four days post infection (dpi), a rusty black infection appeared on the plants. Fifteen dpi, the whole leaf of inoculated stem showed typical symptoms, from which the fungus was re-isolated and colonies were maintained in PDA for morphological characterization, which were confirmed as C. radicicola. All trees from three cultivars showed symptoms with a variable severity from cultivar to another. To our knowledge, this is the first report of black scorch disease caused by T. punctulata in Qatar. This report highlighted the incidence of black scorch disease in Qatar demanding future research study to control the pathogen.