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ACTA PRIMING CAPACITIES OF ENDOPHYTIC METHYLOBACTERIUM SP. ON POTATO (SOLANUM TUBEROSUM L.) Pavlo Ardanov A 613 UNIVERSITATIS OULUENSIS

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OULU 2013 A 613 ACTA Pavlo Ardanov UNIVERSITATIS OULUENSIS A SCIENTIAE RERUM NATURALIUM PRIMING CAPACITIES OF ENDOPHYTIC METHYLOBACTERIUM SP. ON POTATO (SOLANUM TUBEROSUM L.) UNIVERSITY OF OULU GRADUATE
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OULU 2013 A 613 ACTA Pavlo Ardanov UNIVERSITATIS OULUENSIS A SCIENTIAE RERUM NATURALIUM PRIMING CAPACITIES OF ENDOPHYTIC METHYLOBACTERIUM SP. ON POTATO (SOLANUM TUBEROSUM L.) UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF SCIENCE, DEPARTMENT OF BIOLOGY; FINNISH DOCTORAL PROGRAM IN PLANT SCIENCE (FDPPS); INSTITUTE OF MOLECULAR BIOLOGY AND GENETICS OF NATIONAL ACADEMY OF SCIENCE OF UKRAINE ACTA UNIVERSITATIS OULUENSIS A Scientiae Rerum Naturalium 613 PAVLO ARDANOV PRIMING CAPACITIES OF ENDOPHYTIC METHYLOBACTERIUM SP. ON POTATO (SOLANUM TUBEROSUM L.) Academic dissertation to be presented with the assent of the Doctoral Training Committee of Technology and Natural Sciences of the University of Oulu for public defence in Kuusamonsali (Auditorium YB210), Linnanmaa, on 30 August 2013, at 12 noon UNIVERSITY OF OULU, OULU 2013 Copyright 2013 Acta Univ. Oul. A 613, 2013 Supervised by Docent Anna Maria Pirttilä Professor Hely Häggman Doctor Natalia Kozyrovska Reviewed by Professor Rauni Strömmer Doctor Leo Van Overbeek Opponent Docent Minna Pirhonen ISBN (Paperback) ISBN (PDF) ISSN (Printed) ISSN X (Online) Cover Design Raimo Ahonen JUVENES PRINT TAMPERE 2013 Ardanov, Pavlo, Priming capacities of endophytic Methylobacterium sp. on potato (Solanum tuberosum L.) University of Oulu Graduate School; University of Oulu, Faculty of Science, Department of Biology; Finnish Doctoral Program in Plant Science (FDPPS); Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine Acta Univ. Oul. A 613, 2013 University of Oulu, P.O. Box 8000, FI University of Oulu, Finland Abstract The plant can be considered a superorganism that consists of the plant per se and numerous populations of pro- and eukaryotic microorganisms. The interactions between the plant and endophytic microorganisms colonizing plant internal tissues are typically commensalistic or mutualistic. However, information on the role of endophytes in plant defense is limited because pathways are only partly known and systemic responses are typically not seen. The aim of this thesis was to study the priming capacities of endophytic Methylobacterium sp. IMBG290 on potato (Solanum tuberosum L.). Priming of plants by non-pathogenic bacteria allows the host to save energy and to reduce time needed for development of defense reaction during a pathogen attack. Priming phenomenon was demonstrated for Methylobacterium sp. IMBG290 as an activation of salicylic acid and jasmonate/ ethylene-dependent defense pathways after challenge inoculation with the pathogen. Moderate activation of plant antioxidant system may also contribute to resistance induction by the strain. The viable but nonculturable state is presumably a survival strategy observed for the majority of bacterial endophytes. Pathogen attack or environmental changes can activate these quiescent forms. Thus Methylobacterium sp. IMBG290 became cultivable upon plant inoculation by nonpathogenic bacteria. I observed that the composition of the endophyte community changed in response to Methylobacterium sp. IMBG290 inoculation in shoot tissues and correlated with potato disease resistance and growth promotion. Therefore, the activation of endophytic bacterial populations as a putative mechanism of plant disease resistance was proposed. Endophytes have a high agricultural potential. Growth- and resistance-promoting capacities of Methylobacterium sp. IMBG290 on potato were highly variable depending on the cultivar, pathogen, inoculum density, and environmental conditions. Context-dependent efficacy requires more attention when designing complex microbial inoculants capable influencing positively plant growth, resistance, and nutritional properties. Keywords: biological control, endophytes, Methylobacterium, microbial community, priming, Solanum tuberosum L., viable but nonculturable state Ardanov, Pavlo, Endofyyttisen Methylobacterium kannan vastustuskykyä lisäävät ominaisuudet perunalla (Solanum tuberosum L.) Oulun yliopiston tutkijakoulu; Oulun yliopisto, Luonnontieteellinen tiedekunta, Biologian laitos; Kasvitieteen kansallinen tohtoriohjelma (FDPPS); Institute of Molecular Biology and Genetics of National Academy of Science of Ukraine Acta Univ. Oul. A 613, 2013 Oulun yliopisto, PL 8000, Oulun yliopisto Tiivistelmä Kasvia voidaan pitää superorganismina, joka koostuu kasvista itsestään ja lukuisista pro-ja eukaryottisista mikrobipopulaatioista. Kasvin ja sen sisäosia asuttavien endofyyttisten mikro-organismien väliset vuorovaikutukset ovat yleensä kommensalistisia tai mutualistisia. Endofyyttien rooli kasvin puolustuksessa on kuitenkin huonosti tunnettu, koska reitit tiedetään vain osittain eikä järjestelmällisiä vasteita usein havaita. Tämän väitöskirjatyön tavoitteena oli tutkia endofyyttisen Methylobacterium sp. IMBG290-kannan kykyä vahvistaa perunan (Solanum tuberosum L.) puolustusta. Tautia aiheuttamattomat bakteerit kykenevät vahvistamaan kasvien puolustusta, mikä auttaa isäntäkasvia säästämään energiaa ja nopeuttamaan puolustusreaktiota patogeenihyökkäyksen aikana. Methylobacterium sp. IMBG290-kannan vahvistuskyvyn osoitettiin perustuvan salisyylihappo- ja jasmonaatti/etyleeni-riippuvaisten puolustusreittien aktivoimiseen patogeeni-istutuksen jälkeen. Antioksidanttijärjestelmän lievä aktivoituminen voi myös vaikuttaa kannan aiheuttamaan vastustuskyvyn lisääntymiseen. Suurimmalle osalle bakteeriendofyyteistä elävä mutta viljelemätön -olotila on luultavasti selviytymisstrategia. Patogeenihyökkäys tai muutokset ympäristössä voivat aktivoida tällaiset hiljaiset olomuodot. Methylobacterium sp. IMBG290-kanta muuttui viljeltävissä olevaan muotoon kun kasviin istutettiin tautia aiheuttamaton bakteeri. Selvitin, että endofyytti-yhteisön koostumus muuttuu vasteena Methylobacterium sp. IMBG290-kannan istuttamiseen kasvin verson solukoissa, korreloiden lisääntyneen perunan vastustuskyvyn ja kasvun kanssa. Siksi endofyyttisten bakteeripopulaatioiden aktivoitumista esitettiin uutena kasvin puolustusmekanismina. Endofyyteillä on suuret mahdollisuudet maataloudessa. Methylobacterium sp. IMBG290- kannan kasvua ja vastustuskykyä lisäävät ominaisuudet perunalla vaihtelivat lajikkeen, patogeenin, lisätyn bakteeriympin ja ympäristöolosuhteiden mukaan. Suunniteltaessa monimutkaisia bakteeriymppejä kasvien kasvin, vastustuskyvyn ja ravintosisällön lisäämiseksi, täytyy tällainen tilanteesta riippuva tehokkuus ottaa enemmän huomioon. Asiasanat: biologinen torjunta, elävä mutta viljelemätön olomuoto, endofyytit, Methylobacterium, mikrobiyhteisö, puolustuskyvyn vahvistaminen, Solanum tuberosum L. Acknowledgments The present work was carried out at the Department of Biology at the University of Oulu. It was financially supported by the Academy of Finland (grant numbers , , and ), the Centre for International Mobility (CIMO), the European Cooperation in Science and Technology (COST Action FA1103), the Federation of European Microbiological Societies (FEMS), the Finnish Doctoral Program in Plant Science (FDPPS), the Niemi Foundation, the Oulu Province Agricultural Economic Society Foundation, the Scandinavian Plant Physiology Society (SPSS), the Society of Experimental Biology (SEB), the Tauno Tönning Foundation, and by the University of Oulu, which are all gratefully acknowledged. I wish to express my sincere gratitude to my supervisors, Doc. Anna Maria Pirttilä, Prof. Hely Häggman (University of Oulu, Finland), and Dr. Natalia Kozyrovska (Institute of Molecular Biology and Genetics of NASU, Kyiv, Ukraine), for their help with planning research work and discussing the results, support with writing articles, dissertation, and language revision as well as finding research grants and composing the applications. Natalia introduced me to the World of endophytes, which became a fascinating topic of my dissertation. Her innovative ideas formed the core of my research. It was a real pleasure for me to work in the research group finely lead by Anna Maria an unforgettable experience of collaborative atmosphere and research freedom. This work would be impossible without trust, support, and experience, which Anna Maria generously shared with me. I would like to express my warmest acknowledgements to Hely, who revealed me a lot of opportunities and gave many excellent advices. Dr. Leonard Simon van Overbeek (Plant Research International, Wageningen UR, the Netherlands), Prof. Rauni Strömmer, and Dr. Minna Pirhonen (University of Helsinki, Finland) are appreciated for reviewing the thesis. Dr. Anastasiia Aleksandrova (the Hospital for Sick Children, Toronto, Canada) is thanked for revising the language of the thesis. I would like to thank Dr. Olga Podolich, Dr. Iryna Zaets, and M.Sc Leonid Ovcharenko for their help in writing manuscripts for this dissertation as well as to Ms. Tamara Vozniuk and Dr. Inna Skrypkina for their technical assistance (Institute of Molecular Biology and Genetics of NASU, Kyiv, Ukraine). Iryna is appreciated for her help with biochemical methods, and Leonid with phylogenetic analyses. I am grateful to Dr. Sofiya Lyastchenko and to the staff of the Institute for Potato Research of UAAS for their help with carrying on the field experiment. Sofiya is especially acknowledged for her incredible organizational 7 skills to make this experiment happen, answering my numerous questions and concerns as non-specialist in applied research and for her valuable comments for my manuscript. Special thanks to Dr. Angela Sessitsch, Dr. Melanie Kuffner, Marlies Polt, and Alexandra Weilharter for guidance with T-RFLP analysis. Angela is especially appreciated for her critical comments to the results interpretation, and for her valuable advices in experiment setup, which were the great lessons for me in proper scientific practice. Several people at the Department of Biology are gratefully acknowledged for their valuable help. I am extremely grateful for the technical assistance of laboratory amanuensis Tuulikki Pakonen, head laboratory manager Hanna-Liisa Suvilampi as well as laboratory technicians Taina Uusitalo and Tarja Törmänen, who all have kindly helped me with the laboratory work. Thanks to laboratory engineer Niilo Rankka and laboratory technician Matti Rauman for technical help. Office secretaries Sisko Veijola, Marja Liisa Mielikäinen, Ritva Paaso-Dahl, and Erja Vaarala as well as amanuensis Minna Vanhatalo are thanked for their help in practical matters. I thank to Dr. Katja Anttila for teaching me on how to use the confocal microscope, Prof. Jari Oksanen for advices on statistical analysis, and Dr. Katja Karppinen for ascorbic acid detection. I am grateful for extremely useful course for the PhD students organized by Prof. Outi Savolainen and Doc. Laura Kvist. I also wish thank the rest of my workmates in Department of Biology and in Plant Physiology unit for their readiness to help in the lab and to share their free time with me. Warm thanks to Abdul Shakoor, Ahmad Eemaeili Taheri, Prof. Anja Hohtola, Anna-Kaisa Anttila, Dr. Anne Jokela, Catherine Fallecker, Dr. Estelle Dumont, Giacomo Cocetta, Doc. Helmi Kuittinen, Dr. Jaana Vuosku, Jaanika Edesi, Janne Koskimäki, Johanna Pohjanen, Dr. Kaloian Nikolov, Doc. Laura Jaakola, Dr. Marian Sarala, Dr. Marjaana Tahkokorpi, Dr. Mysore Tejesvi, Dr. Pedro Picart Faiget, Sannakajsa Nylund, Dr. Soile Jokipii-Lukkari, Suvi Sutela, and Terttu Kämäräinen-Karppinen for making my life in Oulu happier. I want to address my special thank to the Finnish Doctoral Program in Plant Biology where I belong to, and especially to Karen Sims-Huopaniemi for organizing unique courses with the top researchers for the doctoral students. I also want to thank Dr. Mysore Tejesvi, M.Sc Janne Koskimäki (University of Oulu), and to Dr. Pavlo Gilchuk (who supervised my graduate research in Ukraine) for teaching me molecular biology methods. My sincere thanks to my friends in Oulu Vitalii, Zdravka, Ilya, Antonina Daria, Iryna, and to Laura, Terttu, and all of my volleyball mates for breaking the scientific routine. My best friend Volodymyr is especially acknowledged for 8 chatting on Skype with me almost every day I spent abroad and for entertaining me during my vacations. Finally, I wish to thank to my family for their endless love and care. I thank to my mother Olga who taught me the passion for nature and for her constant care of my health and well-being. I thank to my brother Oleksij for his help with computers. In Kyiv, February 2013 Pavlo Ardanov 9 10 Abbreviations AHL APX ARDRA AsA CAT cv/s. EPSs ET GPOX IMBG290 JA KB LB MS PPFMs Pst QS ROS SA SOD T-RFLP N-acyl-homoserine lactone ascorbate peroxidase amplified ribosomal DNA restriction analysis ascorbic acid catalase cultivar/s exopolysaccharides ethylene guaiacol peroxidase Methylobacterium sp. IMBG290 jasmonate King s B broth Luria-Bertani broth Murashige and Skoog agar medium pink-pigmented facultative methylotrophs Pseudomonas syringae pv. tomato DC3000 quorum sensing reactive oxygen species salicylate superoxide dismutase terminally labeled restriction fragment length polymorphism 11 12 List of original publications The thesis is based on the following publications, which are referred to in the text by their Roman numerals: I Ardanov P, Ovcharenko L, Zaets I, Kozyrovska N & Pirttilä AM (2011) Endophytic bacteria enhancing growth and disease resistance of potato (Solanum tuberosum L.). Biol Control 56(1): II Ardanov P, Sessitsch A, Häggman H, Kozyrovska N & Pirttilä AM (2012) Methylobacterium-induced endophyte community changes correspond with protection of plants against pathogen attack. PLoS ONE 7(10): e III Ardanov P, Podolich O, Zaets I, Pirttilä AM & Kozyrovska N (2013) The role of endophytic bacterial community in plant disease resistance. Manuscript. IV Ardanov P, Lyastchenko S, Karppinen K, Häggman H, Kozyrovska N & Pirttilä AM (2013) Crop yield and disease resistance of potato (Solanum tuberosum L), inoculated with a Methylobacterium sp. are modulated through the resident endophyte community and environmental conditions. Manuscript. The author of this thesis has been the primary author in the original publications (I, II, IV) and contributed equally with Natalia Kozyrovska and Anna Maria Pirttilä to develop the concept and drafting the manuscript III. The author has been responsible for the design of the experiments and carried out the analyses, with the exception of the ascorbic acid detection (IV), handled the data and drafted the manuscripts (I-IV). 13 14 Contents Abstract Tiivistelmä Acknowledgments 7 Abbreviations 11 List of original publications 13 Contents 15 1 Introduction Endophytes Plant colonization Functional role Information processing within the endosphere Environmental plasticity Bacteria in the genus Methylobacterium as a model Microbial inoculants in potato production Aims of the study 29 3 Materials and methods Plant material and culture conditions Microbial strains and culture conditions Methylobacterium inoculation and colonization assays Disease and pest resistance assays Growth promotion assays Enzyme activity assays RNA isolation, cdna synthesis, and Real-time PCR DNA extraction and T-RFLP Sequence analysis Determination of ascorbic acid (AsA) content Data processing and statistical analyses Results Effect of IMBG290 on potato growth and resistance Effect of IMBG290 on the plant antioxidant system Expression of plant genes associated with defense reaction Endophytic microbial community analysis Discussion Methylobacterium and its effect on plant growth and resistance 5.2 Molecular mechanisms of resistance induction by Methylobacterium Activation of innate endophytes as a putative mechanism of plant disease resistance Application of Methylobacterium sp. IMBG290 on potato production Conclusions and future prospects 49 References 51 Original publications 61 16 1 Introduction 1.1 Endophytes The term endophyte was first proposed in 1866 by the German botanist Anton de Bary, describing microorganisms that colonize internal tissues of stems and leaves (de Bary 1866). Since then it has been discovered that plants are naturally associated with microorganisms in various ways. There are two extremes, the mutualistic endosymbiotic interaction (such as the root nodule symbiosis of legumes with rhizobia or the formation of arbuscular mycorrhiza with fungi) and pathogenic interactions. Both require high degree of co-adaptation of the partners. This leads to significant alteration of plant morphology and to development of specific mechanisms for manipulation of plant metabolism (e.g. suppression of defense response by microbial effector proteins) (reviewed by Reinhold-Hurek & Hurek 2011). Therefore the definition of endophyte was later revised to specify that infections with endophytes are asymptomatic, that roots as well as shoots may be colonized, and that an endophyte may not remain an endophyte throughout its life cycle (Porras-Alfaro & Bayman 2011). Additionally, based on their colonization pattern, mycorrhizal fungi with emanating hyphae for nutrient transfer from outside sources are also excluded from endophytes (Chanway 1996). The term endophyte is thus defined by location and does not address the nature of the relationship with the plant, unlike mycorrhiza, which specifies a functional relationship. This broad definition implies that in addition to mutualistic and commensalistic symbionts, endophytes could include latent pathogens, latent saprotrophs, and early stages of colonization by mycorrhizal fungi and rhizobia (Porras-Alfaro & Bayman 2011). For some endophytes, even switching from mutualistic to pathogenic interaction is known (e.g. fungi of the family Clavicipitaceae showing proliferative fungal growth and choking of the host inflorescences due to weakening of the host control during resource mobilization for flowering) (Schardl et al. 2004). Endophytes have been isolated from various organs of different plant species, aboveground tissues of liverworts, hornworts, mosses, lycophytes, equisetopsids, ferns, and spermatophytes from the tropics to the arctic, and from the wild to agricultural ecosystems (Arnold 2007). To date, all plant species studied have been found to be colonized by at least one endophyte (Strobel et al. 2004) either systemically or narrowly restricted in particular plant tissues (White 1987, Arnold 17 & Lutzoni 2007). A milestone in the history of endophyte research was the discovery of the endophytic fungus Neotyphodium coenophialum as the causative organism of fescue toxicosis, a syndrome suffered by cattle fed in pastures of the grass Festuca arundinacea and causing major economic losses (Bacon et al. 1977). The most frequently encountered endophytes are fungi (Staniek et al. 2008), and currently all reported endophytes are fungi or bacteria (including actinomycetes) (Strobel et al. 2004). Fungal endophytes fall into two major categories termed clavicipitaceous endophytes and non-clavicipitaceous endophytes (both belonging to Ascomycetes) based on their phylogeny and life history traits (Rodriguez et al. 2009). Bacterial endophytes mostly belong to Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (Rosenblueth & Martinez-Romero 2006). Evidence of fungal endophytes were found in the fossilized tissues dated 400 million years revealing, that endophyte-plant associations may have evolved from the time higher plants first appeared on the earth (Krings et al. 2007). Therefore endophytes played a long and important role in driving the plant evolution and their functional traits (Friesen 2011). However, endophytes, especially bacterial ones, are much less studied comparing to plant pathogens and endosymbionts, rhizospheric and epiphytic microorganisms (Iniguez et al. 2005, Reinhold-Hurek & Hurek 2011) Plant colonization For establishment of endophytes in different tis
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