The role of cuticle permeability in the defence response of sitiens, an abscisic acid deficient tomato mutant, against the necrotrophic pathogen Botrytis cinerea

Botrytis cinerea, a necrotrophic fungus with broad host range, causes significant losses in tomato greenhouse cultivation every year. We have previously shown that a timely production of hydrogen peroxide (H2O2) is involved in resistance to B. cinerea in the abscisic acid-deficient sitiens tomato mutant. Moreover, it was demonstrated that the fast and localized accumulation of H2O2, before the fungal spore penetration, leads to cell wall fortification in the epidermis, effectively arresting spreading of the pathogen (Asselbergh et al. 2007). To explain the rapid defence response in sitiens, we investigated the cuticle of the mutant, as it makes up the first barrier in the sitiens-B.cinerea interaction. Bessire et al. (2007) recently reported that a more permeable cuticle in an Arabidopsis mutant leads to a strong resistance to B. cinerea. They suggested that an increase in leaf permeability leads to the presence of antifungal compounds on the leaf surface. We compared permeability of wild-type and sitiens cuticle and found that sitiens was more permeable. Therefore, we assessed the correlation between cuticle permeability and resistance/susceptibility response in sitiens and wild-type to the necrotroph B. cinerea. Disease index and permeability index were calculated for leaf discs of the same leaf and the correlation between them was statistically analyzed. The results show that permeability varies in leaves of both genotypes in different developmental stages and that there is a significant linear correlation between permeability and resistance in sitiens, whereas in wild-type, no clear correlation between both characteristics was found. Furthermore, HPLC and mass spectrometry were performed to detect the nature of the compounds in leaf exudates of the two genotypes in a time course after inoculation. Using High Performance Anion Exchange Chromatography with Pulse Amperometric Detection (HPAEC-PAD), we observed one peak in the chromatogram of the infection droplets on sitiens leaf at 4 hpi, rising up in successive time points (8, 12 and 16 hpi), while in wild-type this peak was much less pronounced, particularly in early time points. In the MALDI-TOF (Matrix-Assisted Laser Desorption/Ionisation-Time of Flight) spectra, we were able to identify the presence of the plant defensive saponin alpha-tomatine (1034 m/z) and its B. cinerea detoxified form β1-tomatine (902 m/z). In addition, to determine any effective antifungal activity of the leaf diffusates, we incubated PDB (Potato Dextrose Broth) droplets on the leaf surface of sitiens and wild-type for 18 hours and monitored the germination rate of B. cinerea spores until all the spores were germinated; however, no difference in spore germination was observed. The fungistatic effect of leaf diffusates on hyphal growth of the pathogen was assessed by measuring the length of elongating germ tube at different time points. Clear difference was observed between sitiens-incubated and wild-type-incubated PDBs, showing a suppressed hyphal development in spores growing in sitiens-incubated medium. Moreover, we determined an abnormal hyphal structure in these samples, visible upon the spore germination, using microscopic approach. It seems that the more permeable cuticle in sitiens plant facilitates the release of an antifungal compound, presumably alpha-tomatine, with an efficient fungistatic effect. This hyphal-development suppression might either weaken the pathogenicity or give the plant an extended time to boost its defence reaction