Naima Boughalleb

Effect of dsRNA on growth rate and reproductive potential of Monosporascus cannonballus

The effect of double stranded RNA (dsRNA) infection on growth rate and the reproductive potential of Monosporascus cannonballus was studied in 21 isolates collected in cucurbit growing areas of Spain and Tunisia. The isolates were incubated on potato dextrose agar (PDA) under different conditions of temperature, pH, and water potential (Ψ(s)). They showed optimal growth temperatures over the range of 27-34°C and perithecia formation was obtained mainly at 25 and 30°C, although some isolates were able to produce perithecia at 35°C. All isolates were able to produce perithecia in a broad range of pHs (4-8). Regarding the effect of Ψ(s,) the isolates were more tolerant to grow on KCl than on NaCl. For each solute, radial growth decreased progressively as Ψ(s) decreased and was severely limited at -5.0 to -6.0MPa. Perithecia formation was highest at -0.5MPa, decreased at -1.0MPa and occurred just in some isolates at -2.0MPa. Nine of the M. cannonballus isolates harboured dsRNA with 2-6 bands each and a size range of 1.9-18.0Kb. Phenotypical data were subjected to multivariate factorial analysis. Most of the isolates clustered in two groups corresponding with the presence/absence of dsRNA elements. Isolates without detectable dsRNA produced more perithecia. However, isolates with dsRNA produced lower number of perithecia depending on the pH, Ψ(s,) or solute used. These results improve our understanding of the behaviour and growth of this pathogen in soil, and can be useful to implement effective disease control.

In vitro inhibitory actions of some essential oils on Ascosphaera apis, a fungus responsible for honey bee chalkbrood

Summary Chalkbrood is a highly contagious disease of the honey bee Apis mellifera caused by the heterothallic fungus known as Ascosphaera apis. The disease appears to be most prevalent in the spring when the brood area is increasing. Certain essential oils are known for their antibacterial and antifungal properties. In this study, a number of essential oils have been shown to be particularly effective in controlling the in vitro growth of A. apis. We tested nine essential oils using two in vitro methods, a direct contact method and a confrontation method. The essential oils used were: Lavandula angustifolia; Rosmarinus officinalis; Thymus vulgaris; Salvia officinalis; Mentha x piperita; Pelargonium graveolens; Prunus dulcis; Citrus aurantium; and Olea europaea. Thyme oil produced the best results, followed by the Pelargonium oil. The remaining essential oils were shown to have limited efficacy against A. apis in this in vitro test system, but may contribute to the enhancement of biological or integrated pest management strategies in apiculture.

Monosporascus eutypoides, a Cause of Root Rot and Vine Decline in Tunisia, and Evidence that M. cannonballus and M. eutypoides Are Distinct Species

Three Monosporascus eutypoides-like isolates recovered from cucurbit plants with symptoms of Monosporascus root rot and vine decline in Tunisia were compared to 28 isolates of M. cannonballus from 12 countries for phenotypic, genomic, and pathogenicity characteristics. Morphologically, M. cannonballus and M. eutypoides-like cultures were similar, each producing fertile perithecia in culture containing globose, smooth, dark brown to black ascospores. Nevertheless, all M. cannonballus isolates had one ascospore per ascus, while M. eutypoides-like isolates had mainly two to three ascospores per ascus (rarely one). The employment of the internal transcribed spacer (ITS) of nuclear ribosomal DNA, the elongation factor 1-α (EF-1α), and the β-tubulin (β-tub) gene sequence diversity analyses and the resulting phylogenies identified a level of polymorphism that enabled separation of M. cannonballus and M. eutypoides-like isolates. All isolates of M. cannonballus had identical EF-1α and β-tub sequences irrespective of very diverse geographic origins, which were different from the EF-1α and β-tub sequences of the M. eutypoides-like isolates (96 and 97% similarity, respectively). Similar results were obtained for the ITS region of rDNA. In addition, of three M. eutypoides-like isolates tested for pathogenicity, all three were pathogenic on watermelon, two were pathogenic on muskmelon, but only one was pathogenic on cucumber. The results demonstrate that the M. eutypoides-like isolates belong to the species M. eutypoides, and that M. cannonballus and M. eutypoides are distinct species.

First Report of Gummy Stem Blight Caused by Didymella bryoniae on Grafted Watermelon in Tunisia

During the summer of 2006, severe losses were observed in grafted watermelons in the Testour Region in northern Tunisia. Disease symptoms included stem cankers and necrosis and rot of the grafting area that extended a few centimeters along watermelon vines with the production of a brown gummy exudation. Lesions were not observed on leaves or nongrafted plants. Affected plants wilted and eventually died. The presence of small pseudothecia as black specks was observed embedded in the cankers. Isolations from the stems and crown of symptomatic plants onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate consistently yielded cultures of a fungal agent. These isolates were transferred to PDA and V8 juice agar and incubated at 23°C for 1 month with a 12-h photoperiod. On PDA, they produced numerous pycnidia with hyaline, cylindrical, one-septate conidia, with mean dimensions of 6.7 × 2.5 μm. On V8 juice agar, they produced sparse ostiolate pseudothecia with bitunicate asci and hyaline, oval, one-septate ascospores, with mean dimensions of 13.7 × 5.1 μm. On the basis of these characters, the isolates were identified as Didymella bryoniae (anamorph Phoma cucurbitacearum) (1,2). To further confirm this identification, the complete internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal DNA, of isolates Di-3 and Di-4 were sequenced (GenBank Accession Nos. EF107641 and EF 107642). These sequences were identical to sequences in GenBank from isolates of D. bryoniae (Accession Nos. AF297228 and AF495850). Pathogenicity tests were conducted on watermelon seedlings cv. Giza and Cucurbita hybrid rootstock seedlings cv. Strong Toza using two isolates, Di-3 and Di-4. Seedlings were inoculated at the two- to three-leaf stage. A 5-mm diameter agar disc, cut from the margin of an 8-day-old culture growing on PDA, was inserted in a basal stem wound made with a sterile scalpel at 2 cm above ground level and sealed with Parafilm. Controls were inoculated with sterile PDA discs. There were 10 replicates for each isolate and host with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 10 to 15 days after inoculation, symptoms developed as water-soaked lesions followed by necrosis and finally wilting. The fungus was reisolated from the stems of all inoculated plants, completing Kochs postulates. To our knowledge, this is the first report of D. bryoniae in Tunisia. References: (1) A. P. Keinath et al. Phytopathology 85:364, 1995. (2) E. Punithalingam and P. Holliday. No. 332 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1972.

Saoussanabiloïde, a novel antifungal alkaloid from Echiochilon fruticosum Desf. growing in Tunisia

Bioactivity guided fractionation of Echiochilon fruticosum Desf. (Boraginaceae) butanolic extract biautography assay against fungi led to the isolation of a new bioactive alkaloid, named saoussanabiloïde (1). Its structure was established on the basis of spectroscopic measurements, IR, MS and 2D NMR using COSY, HMQC and HMBC experiments. The strongest inhibitory effect of the butanolic extract, from fractions derived from the crude extract and saoussanabiloïde (1), were observed against Fusarium oxysporum f. sp. niveum.

Physiological races of Fusarium oxysporum f.sp. melonis in Tunisia

Fusarium wilt of melon, caused by Fusarium oxysporum f.sp. melonis (Fom), is an important disease; races of the pathogen were identified by inoculating differential standard host cultivars. A total of ten isolates that were obtained from 23 fields located in four different geographical regions were identified as pathogenic. Results indicate that all four known Fom races, namely, 0, 1, 2 and 1.2, were found in north and middle Tunisia. Race 1.2 was the most prevalent.