Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes.

An enhanced green fluorescent protein has been used to tag an OTA-producing strain of Aspergillus carbonarius (W04-40) isolated from naturally infected grape berries. Transformation of the fungus was mediated by Agrobacterium tumefaciens. The most efficient transformation occurred when the co-cultivation was done with 10(4) conidia due to higher frequency of resistance colonies (894 per 10(4) conidia) and lower background obtained. To confirm the presence of the hph gene in hygromycin resistant colonies, 20 putative transformants were screened by PCR analysis. The hph gene was identified in all the transformants. Variation on the expression levels of the eGFP was detected among the transformants and 50% of them appeared bright green fluorescent under the microscope. Microscopic analysis of all the bright fluorescent transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the conidia. eGFP expression in A. carbonarius was shown to be stable in all transformants. Confocal Laser scanning microscopy images of grape berries infected with the eGFP transformant demonstrated fungal penetration into the berry tissues. OTA production was importantly increased in the eGFP transformant in comparison with the wild type strain and pathogenicity on grape berries was slightly decreased after four days of inoculation. However, no differences in virulence were found after seven days of inoculation, thus allowing utilization of this eGFP mutant for in situ analysis of A. carbonarius infection of grape berries. To our knowledge, this is the first report describing the construction of a GFP-tagged strain belonging to Aspergillus section Nigri for monitoring Aspergillus rot on grape berries.