Chemical interactions between Verticillium longisporum and oilseed rape Brassica napus Dissertation to obtain the Ph. D. degree in the International Ph. D. Program for Agricultural Sciences in Goettingen (IPAG) at the Faculty of Agricultural Sciences, Georg-August-University Göttingen, Germany presented by Husam Ibrahem Aroud born in Damascus, Syria Göttingen, May 2013 D7 1. Name of supervisor: Prof. Dr. Petr Karlovsky 2. Name of co-supervisor: Prof. Dr. Andreas von Tiedemann Date of dissertation: 03.07.2013 Content Table of Contents Chapter 1: General Introduction.........................................................................................7 Oilseed rape Brassica napus.............................................................................................7 Verticillium spp...................................................................................................................8 Verticillium longisporum...............................................................................................10 Interactions between V. longisporum and B. napus........................................................13 Secondary metabolites....................................................................................................15 Plant secondary metabolites.......................................................................................15 Phytoalexins............................................................................................................16 Fungal secondary metabolites....................................................................................19 Polyketides..............................................................................................................20 Aim of the work................................................................................................................21 References.......................................................................................................................22 Chapter 2: Isolation and purification of secondary metabolites from xylem sap of Brassica napus infected with Verticillium longisporum................................................31 Abstract............................................................................................................................31 Introduction......................................................................................................................32 Aim of the work................................................................................................................34 Authors' contributions......................................................................................................35 Materials and Methods....................................................................................................35 Fungal isolate of V. longisporum.................................................................................35 Brassica napus plants.................................................................................................36 Preparation of Verticillium longisporum inoculum.......................................................36 Inoculation of the seedlings with Verticillium longisporum..........................................36 Xylem sap collection....................................................................................................37 High performance liquid chromatography and mass spectrometry............................37 Preparation of xylem sap for purification....................................................................38 Dry weight of xylem sap..............................................................................................38 Purification...................................................................................................................39 First step of purification with preparative HPLC......................................................39 UPLC combined with PDA-TOF MS Detection.......................................................40 Second step of purification (Semi-preparative purification)....................................41 Results.............................................................................................................................42 Content Solubility of xylem sap.................................................................................................42 First step of purification...............................................................................................43 UPLC combined with PDA-TOF- MS Detection of pooled fractions (Sample P)....48 Second step of purification (Semi-preparative purification)........................................50 Discussion........................................................................................................................52 References.......................................................................................................................55 Chapter 3: The effect of Verticillium longisporum infection on the growth of Agrobacterium tumefaciens and Saccharomyces cerevisiae in xylem sap of Brassica napus...................................................................................................................58 Abstract............................................................................................................................58 Introduction......................................................................................................................59 Aim of the work................................................................................................................61 Authors' contributions......................................................................................................61 Materials and Methods....................................................................................................62 Xylem sap....................................................................................................................62 Unfiltered xylem sap................................................................................................62 Brassica napus plants.........................................................................................62 Verticillium longisporum isolate 43.....................................................................62 Preparation of Verticillium longisporum inoculum...............................................63 Inoculating and growing the plants.....................................................................63 Xylem sap extraction...........................................................................................63 Macromolecules filtered xylem sap.........................................................................63 Microbial isolates and media.......................................................................................64 Microbial isolate.......................................................................................................64 LB Medium..............................................................................................................64 YPD Medium...........................................................................................................64 SXM medium...........................................................................................................64 Moisture chamber........................................................................................................65 Inoculum of the bacteria and yeast.........................................................................65 Plating......................................................................................................................66 Treatments..............................................................................................................66 Counting of the colonies..........................................................................................66 Statistical analysis and graphs................................................................................66 Results.............................................................................................................................67 Content Bioassay on the bacteria Agrobacterium tumefaciens................................................67 The unfiltered xylem sap.........................................................................................67 The macromolecules-filtered xylem sap.................................................................69 Bioassay on the yeast Saccharomyces cerevisiae.....................................................69 The unfiltered xylem sap.........................................................................................69 The macromolecules-filtered xylem sap.................................................................72 Discussion........................................................................................................................73 References.......................................................................................................................75 Chapter 4: Comparative metabolic profiling of wild type and polyketide synthase silenced strain of Verticillium longisporum....................................................................79 Abstract............................................................................................................................79 Introduction......................................................................................................................80