Taxonomical study of predatory and plant-parasitic mites associated with South African Solanaceae Candice Ceustermans 21707839 Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in Zoology at the Potchefstroom Campus of the North-West University Supervisor: Prof S Barnard Co-supervisor: Prof EA Ueckermann Assistant Supervisor: Dr LR Tiedt May 2015 i ACKNOWLEDGEMENTS I would like to thank my supervisor, Professor Sandra Barnard (NWU, Potchefstroom), for her guidance, help, support and critical analysis throughout my Masters studies. Also, I would like to thank her for allowing me to collect data on her farm outside of Potchefstroom. I thank my co-supervisor, Professor Eddie Ueckermann (ARC-LNR Roodeplaat, Pretoria), for his support and valuable comments during the study. I also want to thank him for his input and encouragement, as well as the supply of material, his assistance in indentifying mite species and for expanding my thinking about mites. My appreciation goes to my assistant supervisor, Dr. Louwrens Tiedt (NWU, Potchefstroom), for supplying funding for the study and his assistance on the scanning electron microscope. I also thank him for his input during the study and for the use of the laboratory for electron microscopy. My appreciation goes to Mr. Stanley Rens for granting me permission for collecting species on his game farm in Dinokeng, also to Dinokeng Game Reserve for permitting me to collect mite samples on solanaceous plants on their grounds. My appreciation goes to Louis Botha Children's Home for allowing me to collect data on their plot, inni Koppie, in Kameelfontein. I am grateful to Hennie and Hannetjie Barnard at Castello Farming for permitting me to collect mite samples on their farm. A great many thanks to the School of Environmental Science, North-West University, Potchefstroom campus for providing laboratory facilities. Lastly, I would like to thank Ankara University, Plant Protection Department (Turkey) for the opportunity to do this project and to everyone who has contributed to the important knowledge I have gained from the study. ________________________________________ i NORTH-WEST UNIVERSITY Mites on Solanaceae PROJECT COLLABORATION This project is part of a collaborative project (Table 1) supported by three universities [Ankara University (AU) and Uludağ University (UU) in Turkey, and North-West University (NWU) in South Africa] and two institutes [French National Institute for Agricultural Research (INRA), the Centre for Biology and Management of Populations (CBGP) in Montpellier France, and Agricultural Research Council, Plant Protection Research Institute (ARC-PPRI) in Roodeplaat South Africa] that will enable integration of all discrimination methods. This exchange project will enable the building of capacity through the preparation of young scientists about identification of common plant parasitic and predatory mite species using new (molecular and SEM) together with classical methods. The proposed exchange programme is considered to be an opportunity of developing new discriminating tools for pest and predatory mite species with knowledge transfer among European (Turkish and French) and South African scientists. This project carries out a clear application and enables the transfer of expertise from science into practice. Full Title of Collaborative Project: Detection and analysis of inter- and intra-specific variability of common pest and predatory mites using new molecular and imaging tools. Table 1. Partner List Partner Number Partner Name Partner Short Name Country 1 Co-ordinator Ankara University, Plant AU Turkey Protection Department 2 Partner Uludağ University, Plant UU Turkey Protection Department 3 Partner French National Institute for INRA France Agricultural Research, the Centre for Biological and Management of Populations 4 Partner Agricultural Research Council, ARC South Africa Plant Protection Research Institute 5 Partner North-West University NWU South Africa ii NORTH-WEST UNIVERSITY Mites on Solanaceae ABSTRACT Plant-feeding mites represent major pests in agriculture that are of importance to crops world- wide, as large populations of mites reduce the quality and quantity of yields. Alternatives to the use of pesticides are needed due to their negative effects and bio-control agents (predatory mites) remain advantages as they suppress spider mites and other plant pests. This study aims to determine species status of plant-feeding and predatory mites on plants of the family Solanaceae and to apply morphological and molecular data to determine phylogenetic relationships among economically important Phytoseiidae, Stigmaeidae and Tetranychidae. The material for this study was collected through plant beating and specimens were preserved in 75% and 96% ethanol respectively and mounted in Heinz’s PVA medium on microscope slides. A survey was conducted during peak seasons to provide enough samples of pest and predatory species. Morphological analysis was performed and initial results indicate that 94% of the species identified were parasitic and 6% were predatory, which led to a predator:prey ratio of 1:17, where Tetranychus evansi Baker & Pritchard had the highest frequency of appearance. A modified Qiagen DNeasy tissue kit extraction protocol was used and Polymerase Chain Reaction was performed to amplify ribosomal ITS and mitochondrial COI gene fragments. The nucleotide sequence of a 700-bp fragment for ITS was determined by direct sequencing as well as for a 700-bp and 800-bp fragments for COI. The resulting data included 4 isolates that corresponded morphologically and molecularly with Phytoseiidae and 10 with Stigmaeidae. The phylogenetic trees agreed with the morphological data. For species that lack morphological descriptions in GenBank and are not placed within expected clades, one has to accept the possibility of miss identification and highlights the need to combine morphological and molecular approaches to guarantee solid species diagnosis. Ultimately, Solanaceae contain various parasitic mites, but predators seem low in numbers. This could be problamatic in finding effective bio-control agents. Key words: Spider Mites, Predaceous Mites, Species identification, Tetranychidae, Phytoseidae, Stigmaeidae, Internal Transcribed Spacers (ITS), Cytocrome c Oxidase I (COI), Phylogenetic relationships. ________________________________________ iii NORTH-WEST UNIVERSITY Mites on Solanaceae OPSOMMING Plantvoedende myte verteenwoordig plae in landbou wat belangrik is vir gewasse wêreldwyd, aangesien myte die kwaliteit en kwantiteit van die opbrengs kan verlaag. Alternatiewe vir plaagdoders is nodig as gevolg van hul negatiewe invloede; en biologiese beheermetodes (predatoriese myte) bly voordelig aangesien hulle spinmyte en ander peste onderdruk. Die studie se doelwit was om die status van plantvoedende en predatoriese spesies op Solanaceae vas te stel en morfologiese en molekulêre data te gebruik om filogenetiese verwantskappe tussen ekonomies belangrike Phytoseiidae, Stigmaeidae en Tetranychidae te bepaal. Die studiemateriaal is versamel deur plantklopping. Monsters is bewaar in 75% en 96% etanol onderskeidelik en gemonteer in Heinz se PVA-medium op mikroskoopplaatjies. Opnames is tydens piekseisoene gedoen om te verseker dat genoeg parasitiese en roofmyte versamel word. Morfologiese analises dui aan dat 94% van die geïdentifiseerde spesies parasities was en 6% predatories. Die bévinding het gelei tot 'n roofmyt:plaag verhouding van 1:17, waarvan Tetranychus evansi Baker & Pritchard die hoogste voorkomsfrekwensie gehad het. 'n Aangepaste ekstraheringsmetode met die Qiagen DNeasy weefselstel is gebruik en daarna die polimerase kettingreaksie om die ribosomale ITS en die mitochondriale COI geenfragmente te isoleer en te vermeerder. Die nukleotiedvolgorde van ʼn 700-bp fragment van die ITS geenfragment is bepaal deur direkte volgordebepaling en 700-bp en 800-bp vir die COI geenfragment. Die gevolglike data sluit 4 isolate wat morfologies en molekulêr ooreengestem het met die Phytoseiidae en 10 met die Stigmaeidae in. Die filogenetiese bome het ooreengestem met die morfologiese data. Spesies gelys in GenBank, waarvan geen morfologiese beskrywings by gedoen is nie en nie in verwagte klades geplaas kan word nie, is moontlik misgeïdentifiseer en dit beklemtoon die noodsaaklikheid om morfologiese en molekulêre benaderings te kombineer om korrekte spesiesdiagnoses te verseker. Solanaceae bevat 'n verskeidenheid plantvoedende myte, maar predatore is grotendeels afwesig. Dit kan problematies wees vir die soektog na effektiewe biologiese beheermetodes. Sleutelwoorde: Spinmyte, Predatoriese myte, Identifisering van spesies, Tetranychidae, Phytoseidae, Stigmaeidae, Interne getranskribeerde gaping DNS (ITS), Sitochroom c oksidase I (COI), Filogenetiese verwantskappe. ________________________________________ iv NORTH-WEST UNIVERSITY Mites on Solanaceae TABLE OF CONTENTS ACKNOWLEDGEMENTS........................................................................................................i PROJECT COLLABORATION................................................................................................ii ABSTRACT..............................................................................................................................iii OPSOMMING..........................................................................................................................iv LIST OF TABLES...................................................................................................................vii