Effects of Phytotoxic Nonenolides, Stagonolide A and Herbarumin I, on Physiological and Biochemical Processes in Leaves and Roots of Sensitive Plants.

Phytotoxic macrolides attract attention as prototypes of new herbicides. However, their mechanisms of action (MOA) on plants have not yet been elucidated. This study addresses the effects of two ten-membered lactones, stagonolide A (STA) and herbarumin I (HBI) produced by the fungus Stagonospora cirsii, on Cirsium arvense, Arabidopsis thaliana and Allium cepa. Bioassay of STA and HBI on punctured leaf discs of C. arvense and A. thaliana was conducted at a concentration of 2 mg/mL to evaluate phenotypic responses, the content of pigments, electrolyte leakage from leaf discs, the level of reactive oxygen species, Hill reaction rate, and the relative rise in chlorophyll a fluorescence. The toxin treatments resulted in necrotic and bleached leaf lesions in the dark and in the light, respectively. In the light, HBI treatment caused the drop of carotenoids content in leaves on both plants. The electrolyte leakage caused by HBI was light-dependent, in contrast with that caused by STA. Both compounds induced light-independent peroxide generation in leaf cells but did not affect photosynthesis 6 h after treatment. STA (10 µg/mL) caused strong disorders in root cells of A. thaliana leading to the complete dissipation of the mitochondrial membrane potential one hour post treatment, as well as DNA fragmentation and disappearance of acidic vesicles in the division zone after 8 h; the effects of HBI (50 µg/mL) were much milder. Furthermore, STA was found to inhibit mitosis but did not affect the cytoskeleton in cells of root tips of A. cepa and C. arvense, respectively. Finally, STA was supposed to inhibit the intracellular vesicular traffic from the endoplasmic reticulum to the Golgi apparatus, thus interfering with mitosis. HBI is likely to have another main MOA, probably inhibiting the biosynthesis of carotenoids.

Prevalence of Beauveria pseudobassiana among entomopathogenic fungi isolated from the hard tick, Ixodes ricinus.

Human and animal disease-transmitting hard ticks (Acari: Ixodidae) are of great concern for public health and animal farming. Alternatives to tick control by chemical acaricides are urgently needed, and one intensively evaluated biocontrol strategy is based on the use of tick-pathogenic filamentous fungi. An indispensable prerequisite for the development of tick-derived fungal isolates into registered myco-acaricides is their sound taxonomic characterisation. A set of fungal strains isolated from ixodid ticks in the Republic of Moldova was genetically characterised at the genus and species level together with further tick-derived fungal isolates from different geographic locations in Europe and North America. In a previous study, the same isolates had been assigned to the species Beauveria bassiana. Using a recent molecular taxonomic approach based on phylogenetic reconstruction from both internal transcribed spacer (ITS) and protein-encoding gene sequences, all fungi investigated were conclusively assigned to one of the two "hyphomycete" genera, Beauveria or Isaria (Ascomycota; Hypocreales; Cordycipitaceae). Within the genus Isaria, two species, Isaria farinosa and Isaria fumosorosea, were equally represented. Within the genus Beauveria, the comparatively rare species Beauveria pseudobassiana was found to strongly prevail among the isolates from Moldova, and one of the two tick-derived Beauveria strains from North America could be assigned to this species as well. In particular, the previous classification as B. bassiana could not be confirmed for any of the characterised tick pathogens from Europe and North America. The data presented here lend support to the hypothesis that within the genus Beauveria specific adaptation to ticks might have occurred within the species B. pseudobassiana. To test this hypothesis, a more extensive molecular taxonomic survey carefully reconsidering previous taxonomic assignments of tick-derived fungal isolates is needed.

Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana.

Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10(-4) and suppression of background growth were achieved. Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations. Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach. When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement. Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.