Hochschule Geisenheim University and Justus-Liebig-Universität Gießen Institut für Phytopathologie FB09 – Agrarwissenschaften, Ökotrophologie und Umweltmanagement Diagnosis, Transmission, and Management of Phytoplasmas Infecting Rubus Species Submitted in fulfilment of the requirements for the degree Doktor der Agrarwissenschaften (Dr. agr.) Submitted by Holger Linck, M. Sc. Born: 26 July 1986 Ravensburg, Germany Geisenheim, January 2019 This thesis was accepted on 08 July 2019 as a doctoral dissertation/thesis in fulfilment of the requirements for the degree Doktor der Agrarwissenschaften (Dr. agr.) by the Hochschule Geisenheim University and the Justus-Liebig-Universität Gießen. Examination Committee Supervisor and 1st Reviewer: Prof.Dr. Annette Reineke Supervisor and 2nd Reviewer: Prof.Dr. Karl-Heinz Kogel 3rd Reviewer: Prof. Dr. Ralf T. Vögele Examiner: Prof. Dr. Heiko Mibus-Schoppe Examiner: Dr. Erika Krüger-Steden Head of the Committee: Prof. Dr. Claudia Kammann Parts of this thesis have been published in peer-reviewed journals as: Linck, Holger, Erika Krüger, and Annette Reineke. 2017. "A Multiplex TaqMan qPCR Assay for Sensitive and Rapid Detection of Phytoplasmas Infecting Rubus Species". PLoS ONE 12(5):e0177808. Linck, Holger and Annette Reineke. 2019. "Preliminary Survey on Putative Insect Vectors for Rubus Stunt Phytoplasmas". Journal of Applied Entomology 143(4): 328-332. Linck, Holger, Christa Lankes, Erika Krüger, Annette Reineke. 2019. "Elimination of Phytoplasmas in Rubus Mother Plants by Tissue Culture Coupled with Heat Therapy". Plant Disease 103(6): 1252-1255. Linck, Holger, and Annette Reineke. 2019. "Rubus stunt: a review of an important phytoplasma disease in Rubus spp.". Journal of Plant Diseases and Protection. https://doi.org/10.1007/s41348-019-00247-3 I Contents List of Abbreviations ........................................................................................... IV List of Figures ...................................................................................................... VI List of Tables ....................................................................................................... XI 1 Summary ........................................................................................................ 1 2 Zusammenfassung ....................................................................................... 3 3 General Introduction ..................................................................................... 5 3.1 Phytoplasmas .......................................................................................... 6 3.1.1 Morphology, Symptoms, Genomics, and Taxonomy ...................... 6 3.1.2 Transmission and Control ............................................................ 13 3.2 Rubus stunt ............................................................................................ 16 3.2.1 History, Geographic Distribution, and Phytoplasma Agents ......... 16 3.2.2 Symptoms and Transmission ....................................................... 17 3.2.3 Economic Importance and Control ............................................... 21 3.3 Molecular Plant Disease Detection ........................................................ 22 3.4 Aim of this Thesis ................................................................................... 25 4 A Multiplex TaqMan qPCR Assay for Sensitive and Rapid Detection of Phytoplasmas Causing Rubus Stunt ........................................................ 26 4.1 Abstract .................................................................................................. 27 4.2 Introduction ............................................................................................ 27 4.3 Materials and Methods ........................................................................... 29 4.3.1 Plant Material and Plant DNA Extraction ..................................... 29 4.3.2 Insect Samples and Insect DNA Extraction ................................. 33 4.3.3 Oligonucleotide Primers and Probes ............................................ 33 4.3.4 Standard Curve ............................................................................ 37 II 4.3.5 TaqMan qPCR Assay .................................................................. 38 4.3.6 Nested PCR ................................................................................. 39 4.4 Results ................................................................................................... 39 4.4.1 Standard Curve ............................................................................ 39 4.4.2 Validation of the Multiplex TaqMan Assay for Plant Material ....... 41 4.4.3 Validation of the Multiplex TaqMan Assay for Phytoplasmas Present in Insects ........................................................................ 45 4.4.4 Field Validation of the Multiplex TaqMan Assay .......................... 47 4.5 Discussion .............................................................................................. 48 5 Screening of Putative Insect Vectors of Rubus Stunt Phytoplasmas .... 53 5.1 Introduction ............................................................................................ 54 5.2 Materials and Methods ........................................................................... 55 5.2.1 Sampling of Insects ...................................................................... 55 5.2.2 Insect DNA Extraction and Phytoplasma DNA Detection ............. 58 5.3 Results ................................................................................................... 59 5.4 Discussion .............................................................................................. 59 6 Propagation of Healthy Planting Material ................................................. 68 6.1 Susceptibility of Different Raspberry Cultivars to Rubus stunt after Artificial Graft Inoculation ....................................................................... 68 6.1.1 Introduction .................................................................................. 68 6.1.2 Materials and Methods ................................................................. 70 6.1.3 Results ......................................................................................... 73 6.1.4 Discussion ................................................................................... 77 6.2 Elimination of Phytoplasmas in Rubus Mother Plants by Tissue Culture Coupled with Heat Therapy .................................................................... 80 6.2.1 Abstract ........................................................................................ 81 6.2.2 Introduction .................................................................................. 81 6.2.3 Materials and Methods ................................................................. 83 6.2.4 Results ......................................................................................... 90 6.2.5 Discussion ................................................................................... 91 III 7 General Conclusion .................................................................................... 94 8 References ................................................................................................... 98 9 Acknowledgments .................................................................................... 118 10 Funding ...................................................................................................... 120 11 Statutory Declaration ................................................................................ 121 IV List of Abbreviations % percent °C degree Celsius µl microliter µmol micromole bp base pair Ca. Candidatus CC Creative Commons license cf. confer/conferatur, meaning compare cm centimeter Cq quantification cycle CTAB hexadecyl-trimethyl-ammonium bromide Cy5 Cyanine 5 d day dd.mm.yyyy date as two-digit day followed by two-digit month followed by four-digit year DNA deoxyribonucleic acid E East EDTA ethylenediaminetetraacetic acid EPPO European and Mediterranean Plant Protection Organization FAM fluorescein amidite FAOSTAT Food and Agriculture Organization Corporate Statistical Database g gram ha hectare kb kilo base pairs kg kilogram l liter LAMP loop-mediated isothermal amplification L x W x H length by width by height m meter M molar (moles per liter) V min minute ml milliliter MLOs mycoplasma-like organisms mM millimolar mm millimeter mol% mole percent N North NaCl sodium chloride ng nanogram NGS Next-Generation Sequencing nm nanometer No. number(s) NTC no-template control PCR polymerase chain reaction pH decimal logarithm of the reciprocal of the hydrogen ion activity qPCR quantitative polymerase chain reaction R2 coefficient of determination RFLP restriction fragment length polymorphism RNA ribonucleic acid ROX rhodamine X rpm revolutions per minute s second SD standard deviation sec second sp. species (singular) spp. species (plural) Tris-HCl 2-Amino-2-(hydroxymethyl)propane-1,3-diol hydrochloride UV ultraviolet V volt VI List of Figures Figure 1: Transmission electron microscopy micrographs of phytoplasmas floating in the sieve element (SE) lumen. (A, B) Phytoplasmas are mostly roundish, sometimes elongated; a few are dividing (black arrows). (C) Aggregates of SE actin form unipolar fields on the phytoplasma surface in the SE lumen (white arrow). The arrowhead in (B) indicates the attachment of a phytoplasma to the SE plasma membrane. In (A), the