• pathogenicity test
• Lupinus spp.
• anthracnosis
• Acutatum species complex
• phylogenesis

The genus Lupinus, a member of Fabaceae family, includes numerous both annual and perennial species that are cultivated for three main reasons: i) for human nutrition due to their high protein and oil contents; ii) for their contribution to improve soil structure, with an increase of the organic matter content and nitrogen and phosphorus accumulation and, iii) to assure a protein supplement in animal feeds.
The most important threat affecting lupins is anthracnosis caused by Colletotrichum lupini, a seed- and air-borne pathogen infecting crops in the early stage causing a heavy germination and seedling emergence reduction. The symptoms on emerged plants consist in curling of stems, coupled with necrosis leading to the death of the plants even at an early development stage. The disease is currently present in almost every area where lupin is cultivated, including North and South America, Europe and South Africa.
C. lupini belongs to C. acutatum species complex including 34 species generally with a wide host range. Although the C. acutatum species complex is regarded as polyphagous, the causal agent of lupin anthracnose has been shown to form a well-defined, homogeneous and host-specific group. Previous studies performed on C. lupini genome compared with those of other species included in the Acutatum species complex hypothesized a possible involvement, in the pathogenic mechanisms occurring during the interaction between C. lupini and Lupinus sp., of a PKS-NRPS hybrid cluster positioned within a species-specific region,
With the purpose to deeply investigate the role of this cluster in the host preference between C. lupini and Lupinus sp. this work was finalized to acquire more information on the LSR in which the PKS-NRPS1 cluster is present, by understanding its structure, function and the evolutionary origin. Bioinformatic analysis performed during the first part of the present thesis demonstrated that the structure of the cluster seems to be the result of a series of events of different nature such as a horizontal gene transfer of the PKS-NRPS hybrid gene from Penicillium genus, as well as the result of insertion of transposable elements and of a strong selective pressure at the level of the region.
In the second part of the work, two mutants of the C. lupini RB221 isolate (RB221 alfa and RB221 beta), previously obtained by OSCAR protocol with the aim to inactivate the expression of the PKS-NRPS1 cluster, have been stabilized and characterized. In both the mutated strains, the success of the deletion of the portion codifying the start codon methionine and the KS domain in PKS-NRPS hybrid gene has been demonstrated by different PCR after the construction of specific primers. Once confirmed the success of the transformation, the mutants were used for artificial inoculation of L. albus seeds, in order to evaluate the difference in their pathogenicity compared with that of the wt and with a C. panamense isolate, not specific for Lupins. Results obtained from pathogenicity tests confirmed the host specificity of C. lupini compared with C. panamense and showed two different levels of pathogenicity for the two KS mutants. In details, RB221 alfa showed the same aggressiveness of the wt in terms of plant emergence and of plants affected, at cotyledons stage, by antrachnose, whereas RB221 beta affected a significant lower number of plantlets, before foliar stages, when used to inoculate lupins seeds.
What obtained in the present thesis represents only the starting point for future studies where expression analysis of genes in wild-type and mutant strains will be investigated as well as the analysis of secondary metabolites of the wt compared with those of the two mutants in order to better investigate the role of PKS-NRPS1 in host specificity between C. lupini and Lupinus spp.