Alternaria blackspot phytotoxins : biotransformation and phytoalexin elicitation

Abstract

Alternaria blackspot, one of the most destructive diseases affecting the 'Brassica' species worldwide, is caused by the fungus ' Alternaria brassicae' (Berkeley) Saccardo. The pathogen produces destruxin B and homodestruxin B, two important host-selective phytotoxins. Studies on their biotransformation by cruciferous species susceptible ('Brassica napus, Brassica juncea') and resistant ('Sinapis alba') to alternaria blackspot were undertaken. The phytotoxin metabolism was studied by monitoring the fate of 14C-labeled destruxins in leaf tissue. Leaves were fed with a solution of 14C-labeled toxin, incubated for different periods, then extracted with organic solvents. Leaf extracts were analyzed by HPLC and LSC. Scale-up experiments using non-labeled destruxins, followed by FCC and prepTLC resulted in the isolation of the metabolites which were characterized by spectroscopic techniques (1H, 13C NMR, MS, FTIR). Bioassays were conducted to compare the phytotoxic activity of the parent compound and its metabolite. Leaves of blackspot resistant and susceptible crucifer species biotransformed destruxin B to hydroxydestruxin B. Similarly, homodestruxin B was metabolized to hydroxyhomodestruxin B. The rate of these transformations (higher in the resistant species than in the susceptible ones) correlated with the resistance and susceptibility of these species to 'Alternaria brassicae'. Further biotransformation experiments of hydroxydestruxin B by the same resistant ('S. alba') and susceptible ('B. napus, B. juncea') species showed its transformation to β-D-glucosyl hydroxydestruxin B, faster in the susceptible species. Overall, results of these studies showed that metabolism of destruxin B and homodestruxin B occurring in cruciferous resistant species were detoxification processes. Also, leaves of blackspot resistant 'Camelina sativa' (cruciferous weed) metabolized destruxin B to β-D-glucosyl hydroxydestruxin B. Elicitation of phytoalexins in 'S. alba' leaves using destruxins led to discovery of sinalbins A and B. Upon isolation (FCC, prepTLC) and structural elucidation (NMR, MS), their syntheses, and that of sinalexin, a known 'S. alba' phytoalexin, were undertaken. Thus, a new work-up procedure following a classical Vilsmeier formylation was developed, and applied to the synthesis of sinalexin and brassilexin, another important cruciferous phytoalexin. Further, these compounds were bioassayed. Sinalbins A and B showed antifungal activity against 'Phoma lingam', while sinalexin and brassilexin were active against 'A. brassicae, P. lingam, Rhizoctonia solani' and Sclerotinia sclerotiorum fungi.