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 ß-tubulin (ß-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 ß-tub sequences irrespective of very diverse geographic origins, which were different from the EF-1α and ß-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.

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.

Antifungal activity from polar and non-polar extracts of some Chenopodiaceae wild species growing in Tunisia.

Nine plants belonging to Chenopodiaceae family were collected around salt marshes near Monastir, located in the east Mediterranean coast of Tunisia. They were tested for their antifungal activities against six plant pathogenic fungi: Botrytis cinerea, Fusarium oxysporum f. sp. niveum, F. solani f. sp. cucurbitae, Phytophthora cactorum, Rhizoctonia solani and Nattrassia mangiferae. Data of this study showed that the highest inhibition of Botrytis cinerea growth was observed with the petroleum ether extract of Atriplex inflata fruits (F) (24.5 mm). The in vitro growth of F. oxysporum f. sp. niveum was reduced only with A. inflata whole plant (WP) petroleum ether extract (32.3 mm). The most important inhibition zones were obtained against F. solani f. sp. cucurbitae with Atriplex semibaccata methanol and acetone extracts (34.7 and 31.0 mm, respectively). This work revealed that fungitoxic compounds were probably present in the petroleum ether extract obtained from A. portulacoides (WP), since it has suppressed the growth of F. s. cucurbitae. Our investigation proved that many Chenopodiaceae species adapted to saline soils may contain phytochemical compounds with fungicidal properties.

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 Koch's 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.