First Report of Thielaviopsis punctulata Causing Black Scorch Disease on Date Palm in Qatar.

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. References: (1) Y. M. Al-Raisi et al. New Dis. Rep. 23:23, 2011. (2) C. Linde and W. A. Smit. Plant Dis. 83:880, 1999. (3) P. Suleman et al. Plant Dis. 85:80, 2001.

Resistance of sunflower to the biotrophic oomycete Plasmopara halstedii is associated with a delayed hypersensitive response within the hypocotyls.

The biotrophic oomycete Plasmopara halstedii is the causal agent of downy mildew in sunflower. It penetrates the roots of both susceptible and resistant sunflower lines and grows through the hypocotyls towards the upper part of the seedling. RT-PCR analysis has shown that resistance is associated with the activation of a hsr203J-like gene, which is a molecular marker of the hypersensitive reaction in tobacco. Activation of this gene was specifically observed during the incompatible interaction and coincided with cell collapse in the hypocotyls. This HR was also associated with the early and local activation of the NPR1 gene which is a key component in the establishment of the SAR. No such HR or a significant activation of the hsr203J-like gene were observed during the compatible combination. These results suggest that the resistance of sunflower to P. halstedii is associated with an HR which fails to halt the parasite. By contrast, this HR triggers a SAR which takes places in the upper part of the hypocotyls and eventually leads to the arrest of parasite growth. A model describing the resistance of plants to root-infecting oomycetes is proposed.

Induction of a sunflower CC-NBS-LRR resistance gene analogue during incompatible interaction with Plasmopara halstedii.

Downy mildew caused by Plasmopara halstedii is one of the main diseases causing economic losses in cultivated sunflower. Resistance in this host is conferred by major genes denoted Pl. The inbred sunflower line QIR8, which contains the Pl8 locus and is resistant to all known downy mildew races, was used to isolate a full-length resistance gene analogue (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The genetically incompatible combination involving downy mildew races 300 and sunflower line QIR8 was characterized by a hypersensitive-like reaction. Semi-quantitative RT-PCR analysis showed that the transcript of Ha-NTIR11g RGA was specifically induced during the incompatible reaction. The transcript was induced approximately 3 d post-infection (dpi), and then decreased by 9 dpi. The high level of transcriptional expression of this RGA coincides with a transcript accumulation of the hsr203J gene which is a marker of the hypersensitive reaction. Treatment with signalling molecules, including salicylic acid and methyl jasmonate, did not activate transcription of the Ha-NTIR11g gene, indicating that Ha-NTIR11g expression is not regulated by defence signalling pathways triggered by these molecules. Ha-NTIR11g was not induced by treatment with hydrogen peroxide or wounding. These results suggest that Ha-NTIR11g RGA may play a critical role in protecting sunflower cells against P. halstedii. The transcript accumulation of R gene-mediated signalling components was also examined.

Development of PCR markers for the Pl5/Pl8 locus for resistance to Plasmopara halstedii in sunflower, Helianthus annuus L. from complete CC-NBS-LRR sequences.

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.

Identification of non-TIR-NBS-LRR markers linked to the Pl5/ Pl8 locus for resistance to downy mildew in sunflower.

The resistance of sunflower, Helianthus annuus L., to downy mildew, caused by Plasmopara halstedii, is conferred by major genes denoted by Pl. Using degenerate and specific primers, 16 different resistance gene analogs (RGAs) have been cloned and sequenced. Sequence comparison and Southern-blot analysis distinguished six classes of RGA. Two of these classes correspond to TIR-NBS-LRR sequences while the remaining four classes correspond to the non-TIR-NBS-LRR type of resistance genes. The genetic mapping of these RGAs on two segregating F2 populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked to the Pl5/ Pl8 locus for resistance to downy mildew in sunflower. These and other results indicate that different Pl loci conferring resistance to the same pathogen races may contain different sequences.