Investigation on host specificity of Colletotrichum lupini

Colletotrichum lupini is a plant pathogen responsible for anthracnose disease of lupin, where it causes severe symptoms leading to severe yield losses. C. lupini is a member of the acutatum species complex and, differently from the other members of this complex, it possesses a high specificity for its host, making it an interesting evolutionary model for host-pathogen interaction studies. The aim of this thesis was to investigate the interaction between Colletotrichum lupini and Lupinus albus, in order to determine the host specificity of C. lupini compared to other related species included in the same complex. A pathogenicity test was performed by artificially inoculating pre-germinated L. albus cv Italia seeds. The test was made by using 10 C. lupini isolates together with isolates belonging to other Colletotrichum species, with some of them included in the acutatum species complex. Numbers of emerged plants and of plants showing the typical symptoms of anthracnose (necrosis on hypocotyl), were recorded and submitted to statistical analysis. Results obtained from ANOVA showed that all C. lupini isolates were able to cause an infection rate significantly higher than that obtained by the other isolates, whereas no differences were evident among the different C. lupini isolates. Results confirmed that C. lupini is able to infect more aggressively and more efficiently lupine plants, compared to other species of Colletotrichum. In order to follow the initial stages of the infection (biotrophic phase) of C. lupini on its host, a constitutively expressing GFP (Green Fluorescent Protein) isolate was used to artificially inoculate lupine seeds and plant colonization was monitored under fluorescent microscopy. First observations made at different time after the inoculation allowed to define the time of production of appressoria (after 12 hours from the inoculation), as the starting point of tissue infection . Based on a comparative genomic study, a lineage-specific cluster has been found in C. lupini. In order to verify the involvement of this cluster in the host-specificity, a polyketide-synthase gene included in this cluster was targeted for site-specific knock-out through the use of a new protocol. This protocol, named OSCAR, is an improvement of the Gateway technology and utilises Agrobacterium mediated transformation (AMT) to obtain mutant strains. After the amplification of the flanking regions of the gene of interest, a one-step clonase reaction was performed, thus resulting in new constructs to be used for the knock-out in C. lupini transformation. Results here obtained are the starting point for further analysis aimed to verify the involvement of the selected genes in the host specificity showed by C. lupini on Lupinus spp