Induction of defence responses and resistance to wheat leaf rust by plant extracts

English: Information on the induced disease resistance mechanism in wheat against leaf rust (Puccinia triticina) by two natural bio-stimulants (ComCat® and the seed suspension of Lupinus albus; SS) and two extracts with antifungal activity (Tulbaghia violacea and Agapanthus africanus) may be of great value both in designing new agrochemicals that stimulate plant resistance responses and in developing genetically engineered plants with enhanced disease resistance. The potential of these extracts to control leaf rust in vivo in susceptible (Thatcher) and resistant (Thatcher / Lr15) wheat was investigated. ComCat® and SS had no direct effect while the A. africanus extract resulted in the reduction of pustule and necrotic lesion formation in a susceptible and resistant wheat cultivar. T. violacea and A. africanus significantly inhibited the germination of P. triticina spores and prevented further germ tube development. Foliar application of the different plant extracts on resistant infected wheat plants activated β-1,3-glucanase, chitinase and peroxidase enzyme activities. However, it was only the A. africanus treatment that increased the in vitro activities of these three apoplastic PRproteins significantly in both susceptible and resistant wheat cultivars, whether uninfected or infected. As a result it was decided to concentrate the rest of the study on the A. africanus extract only. The induction pattern of apoplastic proteins from infected susceptible and resistant wheat treated with an A. africanus extract as well as a control treated with distilled water was followed using SDS-PAGE. Clear differences between SDS-PAGE profiles of intercellular proteins from resistant and susceptible as well as untreated and treated plants were observed throughout the 144 h period after treatment with the extract. In general, resistant plants contained higher amounts of a 31 kDa protein and the protein was also present at much higher detectable levels in plants treated with the A. africanus extract. The molecular mass corresponded to that of β-1,3-glucanase. A Western blot using a polyclonal antibody against β-1,3-glucanase from wheat confirmed the identity of the 31 kDa protein to indeed be that of β-1,3-glucanase. This overwhelmingly excluded the A. africanus extract from the rest in terms of its potent ability to induce a defence response in wheat towards leaf rust. RT-PCR was used in the analysis of the expression of the three defence related genes. Timecourse experiments confirmed that they were induced in resistant as well as susceptible wheat after infection. In this study, when resistant and susceptible wheat were treated with an extract of A. africanus 48 h prior to infection, a more pronounced induction of PR2, PR3 and PR9 gene expression occurred. Two different sized fragments were amplified when using PR9 specific primers and both were induced by infection and by treatment with A. africanus extract in susceptible and resistant wheat. After sequencing, the larger fragment was confirmed to be peroxidase, while the smaller fragment shared very high sequence similarity to a retrotransposon gene. It can, therefore, be claimed that A. africanus is responsible for the induction of PR genes and a retrotransposon gene in wheat. From the results obtained thus far, it was obvious that A. africanus must contain an active compound(s) that act as an elicitor(s) in the mechanism of the defence reaction of wheat against leaf rust infection. Subsequently, activity directed isolation and purification of the active compound lead to the isolation of a saponin, identified by means of 1H-NMR and 13CNMR spectroscopy as (25R)- 5α spirostane-2α, 3β, 5α-triol 3-O-{O-α-L-rhamnopyranosyl- (1α2)-O-[β-D-galactopyranosyl- (1α3)]- β-D-glucopyranoside}.