Elimination of Clavibacter michiganensis subsp. michiganensis from tomato cultures and seeds by highly sensitive detection methods and effective seed treatments Dissertation to obtain the PhD degree at the Faculty of Agricultural Sciences, Georg-August-University Göttingen, Germany by Radwan M. Ftayeh born in Deir Al-Bakht Daraa, Syria Göttingen 2009 D7 1. Name of referee: Prof. Dr. Andreas von Tiedemann 2. Name of co-referee: Prof. Dr. Kerstin Wydra 3. Name of co-referee: Prof. Dr. Petr Karlovsky Date of disputation: January 29, 2010 Contents Contents General Introduction ………………………………………………………………….… 1 Disease history …………………………………………………………………..... 1 Tomato production in Germany …………………………………………………... 3 Symptoms ….…………………………………………………………………..…. 4 Disease epidemiolog …………………………………………………………........ 6 Seed health certification ………………………………………………………….. 7 International requirements ………………………………………………………... 8 Objectives ………………………………………………………………………… 9 Outcomes …………………………………………………………………………. 9 References ……………………………………………………………………….. 11 Chapter 1: Development of new selective and highly sensitive nutrient media for Clavibacter michiganensis subsp. michiganensis and other subspecies ……………... 15 Summary ……………………………………………………………………………….. 15 Conclusions …………………………………………………………………………….. 16 Introduction ……………………………………………………………………………. 17 Materials and Methods ………………………………………………………………... 19 Bacterial species and strains …………………………………………………….. 19 Antibiotic-resistant mutant of Cmm ……………………………………………... 20 Media and growth conditions …………………………………………………… 20 Selection of the basic medium for Cmm ………………………………………… 22 Screening of antibiotics …………………………………………………………. 22 Susceptibility of accompanying bacteria towards antibiotics …………………… 23 Adjusting the optimum concentrations of inhibitors ……………………………. 23 Determining the plating efficiency (recovery rate) of Cmm strains on semiselective media …………………...........................…………………………….………….. 24 Evaluation of selectivity and detection sensitivity of semiselective media ……... 24 Results …………………………………………………………………………………... 26 Selecting a new basic medium for Cmm ……………………………………….... 26 Screening of different antibiotics ………………………………………………... 27 Susceptibility of accompanying bacterial species and strains towards antibiotics ……………………………………………………………………...... 29 i Contents Recipes of the new selective media BCT and BCT-2 …………………………… 30 Effect of boric acid………………………………………………………………. 32 Plating efficiency (recovery rate) of Cmm on the published and the new selective media …………...................................…………………………………………... 35 Selectivity of the new media BCT and BCT-2 ………………………………….. 39 Detection sensitivity of the new media for latent infection by Cmm …………… 42 Selectivity for other pathovars/species of coryneform bacteria ………………… 45 Modifications of the new media BCT and BCT-2 ………………………………. 47 Effect of the buffering system and other fungicides …………………………….. 50 Effect of the pH value on Cmm growth …………………………………………. 52 Effect of fruit juice ………………………………………………………………. 53 Selection of a fungicide …………………………...…………………………….. 53 Discussion …………………………………………………...………………………..… 55 References ……………………………………..………………………………………... 60 Chapter 2: Establishment of a Bio-PCR assay for a sensitive detection of Clavibacter michiganensis subsp. michiganensis in seed and plant material ……………………. 65 Summary ……………………………………………………………………………….. 65 Introduction ……………………………………………………………………….…… 67 Materials and Methods ………………………………………………………………... 69 Bacterial cultures and growth conditions ……………………………………….. 69 DNA extraction ………………………………………………………………….. 69 Primer design ……………………………………………………………………. 72 Designing new primers based on the publication of Bach et al. (2003) ………… 72 Designing new primers based on the publication of Luo et. al. (2008) …………. 72 Amplification conditions …................................….........……………………….. 73 - The new primers “B-rev-CM/B-fw-PCM” and “L-fw-CM/L-rev-PCM”; and the primer set tomA-F/tomA-R (Kleitman et al., 2008) …………………………. 73 - The primers CMM-5/CMM-6 (Dreier et al., 1995); CM3/CM4 (Sousa-Santos et al., 1997); and PSA-4/PSA-R (Pastrik and Rainey, 1999) …………...……. 73 Direct PCR ………………………………………………………………………. 75 Inhibitor tests ……………………………………………………………………. 75 Bio-PCR …………………………………………………………………………. 76 Results ………………………………………………………………………………...… 78 Specificity ……………………………………………………………………..… 78 ii Contents Efficiency of the new primers for amplification of different Cmm strains ……… 85 Direct PCR ………………………………………………...…………………….. 85 Inhibitor tests ……………………………………………………………………. 86 Bio-PCR …………………………………………………………………………. 88 Discussion ………………………………………………………………………………. 94 References ……………………………………………………………………………... 101 Chapter 3: Efficacy of different disinfection methods for eradication of Clavibacter michiganensis subsp. michiganensis from tomato seed …………………………….. 107 Summary …………………………………………………………………………….... 107 Introduction …………………………………………………………...……………… 109 Materials and Methods ……………………………………………………...………... 111 Antibiotic-resistant Cmm mutant ………………………………………………. 111 Bacterial inocula and testing of different inoculation methods with Cmm …….. 111 Screening of different chemical and physical seed treatment methods .…......… 113 Seed lots, seed infection and storage conditions ……………………………….. 113 Evaluation of seed germination capacity ………………………………………. 114 Evaluation of seed infection ………………………………………………….... 115 Seed treatments ……………………………………………………………….... 116 Chemical and hot water treatments …………………………………………….. 116 Seed fermentation ……………………………………………………………… 117 Seed extraction with hydrochloric acid ………………………………………... 117 Seed treatments with hot air ……………………………………………………. 118 Statistical analysis ……………………………………………………………… 118 Results …………………………………………………………………………………. 119 Screening of different inoculation methods with Cmm ………………………... 119 Effect of seed treatments towards Cmm bacteria ………………………………. 123 Successful eradication of Cmm from infested seeds by chemical or hot water treatments .................................................................…………………………… 123 Effect of seed fermentation …………………………………………………….. 125 Seed extraction with hydrochloric acid ………………………………………... 125 Effect of hot air treatments …………………………………………………….. 126 Effect of seed treatments on saprophytic bacteria ……………………………... 126 Effect of treatments on seed germination capacity ……………………………...127 Seed germination capacity at two and eight months after treatments ………..... 128 iii Contents Discussion ……………………………………………………………………………... 131 References ……………………………………………………………………………... 135 Chapter 4: Occurrence of Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato, in Syria ……….……………………………..... 139 Summary ………………………………………………………………………….…... 139 Introduction …………………………………………………………………………... 140 Materials and Methods ………………………………………………………………. 141 Surveys and sample collection …………………………………………………. 141 Isolation and identification …………………………………………………….. 141 Pathogenicity …………………………………………………………………... 142 PCR identification ……………………………………………………………... 143 Results …………………………………………………………………………………. 144 Disease incidence ………………………………………………………………. 144 Isolate identification ………………………………………………………….... 144 Pathogenicity …………………………………………………………………... 145 PCR identification ……………………………………………………………... 145 Discussion ……………………………………………………………………………... 147 References ……………………………………………………………………………... 149 General Discussion …………………………………………………………………… 151 General Summary ……………………………………………………………………. 154 Related publications ………………………………………………………………….. 158 Refereed journals ………………………………………………………………. 158 Presentations at national and international conferences ……………………….. 158 Conferences and workshops attended ………………………………………….. 159 Abbreviations …………………………………………………………………………. 160 Acknowledgements ………………………………………………………………….... 162 Eidesstattliche Erklärung ……………………………………………………………. 164 Curriculum vitae ……………………………………………………………………... 165 iv General Introduction General Introduction Disease history Bacterial canker of tomato, caused by the bacterium Clavibacter michiganensis subsp. michiganensis (Smith. 1910) Davis et al. 1984 is a serious and destructive disease worldwide. The disease was at first described by Smith who found it in 1909 in Grand Rapids, Michigan (Strider, 1969), after which the pathogen spread into nearly all main tomato production areas world-wide. Recently, the incidence of bacterial canker of tomato increased in Europe and was newly reported in several countries worldwide causing considerable losses. Therfore, a new distribution map of the pathogen (Figure 1) was issued (CABI/EPPO, 2009). The bacterium is considered as a quarantine organism in the European Union and many other countries (Council Directive 2000/29/EC; OEPP/EPPO, 1982). In Germany, the pathogen is known since 1929 (Kotte, 1930; Stapp, 1958), and caused serious losses in 1978, especially in greenhouses (Griesbach, person. commun.). Recently, the disease was transmitted in 1998 into the peninsula Reichenau in South Germany in Baden- Württemberg (Schmidt, 2006, person. commun.) and newly in 2002 into “Knoblauchsland” near Nürnberg in Bavaria (Maeritz, 2006, personal commun.), also in 2006 into North-Rhine- Westphalia (Matthäus-Staack and Eickeln, 2006, personal commun.) and very recently again into new locations of Baden-Württemberg in 2009 (Moltmann, 2009, personal commun.). Recently, the disease also occurred in neighbouring countries of Germany, such as Austria (Weber and Fuchs, 2007, personal observation and commun.), Switzerland (Wasserfallen,