Abstract
To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples.
Highlights
Monilinia spp. cause brown rot disease, one of the most important diseases on stone and pome fruit [1]
Monilinia fructicola is a single spore isolate collected from a latent infection on a peach in Alfarrás (Lleida, Spain)
Wild type and transformant strains were stored as a conidial suspension in 20% glycerol at −80 ◦ C for long-term storage, and as a culture on potato dextrose agar (PDA; Difco, Detroit, MI, USA) at 4 ◦ C in darkness for short-term storage
Summary
Monilinia spp. cause brown rot disease, one of the most important diseases on stone and pome fruit [1]. M. fructicola (G Winter) Honey and M. laxa Ruhland) Honey are the main species causing brown rot in stone fruit currently in Spain, both with the same frequency after the first detection of M. fructicola in 2006 [3,4]. M. fructigena has been displacing as a minority species on stone fruit [4]. This displacement is supported by the effects of higher growth rate and aggressiveness factors in M. fructicola on stone fruit than M. laxa and. First brown-rot symptoms are visible 24 h post-inoculation on fruit surface and epidermal discoloration
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