Toxin production by Ascochyta rabiei, the causal agent of ascochyta blight of chickpea (Cicer arietinum L.), and development of a transformation protocol for the fungus
Ascochyta rabiei is the causal agent of blight of chickpea, an important food legume crop for human populations in Developing Countries. All reliably identified isolates of the fungus produce toxins in culture, known as the solanapyrones, of which solanapyrone A is the most frequently found and also the most toxic. The principal aim of the project was to determine the role of this toxin in the disease syndrome by producing toxin-minus mutants and testing them for virulence. Four transformation techniques were attempted: Restriction Enzyme Mediated Integration (REMI), electroporation, particle bombardment and Agrobacterium tumefaciens-mediated transformation. With the last, employing a T-DNA containing a hygromycin resistance gene, 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Genuine transformants were tested for the production of solanapyrone A using an assay in microtitre plates. Loss of toxin production by transformants was confirmed by reversed phase High Performance Liquid Chromatography. Sixteen transformants produced significantly less solanapyrone A than the wild-type strain. Transformants were also screened for integration events by PCR, using primers specific to the hygromycin resistance gene and homologous hybridisation to a probe consisting of this gene. Among the four transformants tested, three have integrated two copies of T-DNA and one had a single insertion. In order to optimise the production of solanapyrone A so as to provide a source of the compound for screening chickpea genotypes, three types of cultures of A. rabiei were tested: still culture, shake culture and fermenter culture. The toxin was purified from culture filtrates by solvent partitioning followed by flash chromatography. The effect of two safeners on the sensitivity of chickpea shoots to solanapyrone A was tested using bioassays. Dichlormid (300 or 800 g per shoot) and fenclorim (18 g per shoot) decreased the sensitivity of chickpea shoots to solanapyrone A 1.6 and 2.5-fold, respectively.