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

Abstract

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}.

Afrikaans: Inligting aangaande die biochemiese weerstandsreaksiemeganisme van koring teen blaarroes (Puccinia triticina) geïnduseer deur twee natuurlike bio-stimulante (ComCat® en die saadsuspensie van Lupinus albus; SS), asook twee plantekstrakte met antifungale aktiwiteit (Tulbaghia violacea en Agapanthus africanus), mag van groot belang wees in die ontwikkeling van natuurlike middels asook geneties gemanipuleerde plante met verhoogde weerstand teen siektes, wat aangewend kan word in bestaans en kommersiële boerdery praktyke. Die potensiaal van hierdie ekstrakte om roesweerstand in vivo in vatbare (Thatcher) en weerstandbiedende (Thatcher / Lr15) koring te beheer is ondersoek. ComCat® en SS het geen effek gehad nie, terwyl A. africanus ‘n vermindering in roesontwikkeling in beide kultivars tot gevolg gehad het. A. africanus en T. violacea het ook die ontkieming van roesspore en ontwikkeling van infeksiehifes voorkom. ‘n Blaartoediening van die verskillende ekstrakte op weerstandbiedende geïnfekteerde koringplante het gelei tot verhoogde β-1,3-glukanase, chitinase and peroksidase ensiemaktiwiteite. Dit was egter slegs behandeling met die A. africanus ekstrak wat die in vitro ensiemaktiwiteite van die drie apoplastiese patogeneseverwante (PR) proteïene in beide cultivars, geïnfekteer of ongeïnfekteer, betekenisvol verhoog het. As gevolg van hierdie resultate is besluit om slegs op die A. africanus ekstrak te konsentreer. Deur gebruik te maak van SDS-PAGE jelelektroforese is die akkumulering van apoplastiese proteïene in beide die geïnfekteerde vatbare en weerstandbiedende kultivars, sowel as die wat met met A. africanus ekstrak behandel is, gemonitor. Duidelike verskille in die proteïenprofiele tussen die verskillende cultivars en behandeling met A. africanus ekstrak was waargeneem, met ‘n prominente akkumulasie van ‘n 31 kDa grootte proteïen. Hierdie proteïen is met behulp van Westernkladanalise as β-1,3-glukanase bevestig en dui onteenseglik op die potensiaal van A. africanus om die PR- proteïene en derhalwe die weerstandsrespons van koring teen roesinfeksie te induseer. RT-PCR was gebruik om die uiting van die drie PR gene te bestudeer. Roesinfeksie het tot induksie van die gene gelei, terwyl behandeling van vatbare en weerstandbiedende plante met A. africanus ekstrak ‘n meer prominente induksie van PR2, PR3 en PR9 gedurende die 144 uur periode tot gevolg gehad het. Twee verskillende groottes fragmente is uitgedruk by gebruik van die PR9 spesifieke fragment en albei se uiting was aangeskakel tydens infeksie sowel as behandeling met A. africanus ekstrak in beide vatbare en weerstandbiedende koring. Nadat hul DNA volgorde bepaal is, is die groter fragment geïdentifiseer as peroksidase terwyl die kleiner fragment baie hoë homologie met ‘n retrotransposon geen getoon het. A. africanus is dus verantwoordelik vir die aanskakeling en induksie van PR gene en ‘n retrotransposon geen in koring. Uit die resultate is dit duidelik dat A. africanus ‘n verbinding besit wat as aktiewe bestanddeel dien om die weerstandsreaksie in koring aan te skakel. Gevolglike aktiwiteitsgerigte isolasie en suiwering van die aktiewe komponente betrokke, het aanleiding gegee tot die identifikasie van ‘n saponien, (25R)- 5α spirostaan-2α, 3β, 5α-triol 3-O-{O-α-Lramnopiranosiel-( 1α2)-O-[β-D-galaktopiranosiel- (1α3)]- β-D-glukopiranosied}, met behulp van 1H-KMR and 13C-KMR spektroskopie.