View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Florence Research DOCTORAL THESIS IN Plant, Microbiology and Genetic Science and Technology section of " Plant Protection" (Plant Pathology), Department of Agri-food Production and Environmental Sciences, University of Florence Phytophthora in natural and anthropic environments: new molecular diagnostic tools for early detection and ecological studies Duccio Migliorini Years 2012/2015 DOTTORATO DI RICERCA IN Scienze e Tecnologie Vegetali Microbiologiche e genetiche CICLO XXVIII COORDINATORE Prof. Paolo Capretti Phytophthora in natural and anthropic environments: new molecular diagnostic tools for early detection and ecological studies Settore Scientifico Disciplinare AGR/12 Dottorando Tutore Dott. Duccio Migliorini Dott. Alberto Santini Coordinatore Prof. Paolo Capretti Anni 2012/2015 1 Declaration I hereby declare that this submission is my own work and that, to the best of my knowledge and belief, it contains no material previously published or written by another person nor material which to a substantial extent has been accepted for the award of any other degree or diploma of the university or other institute of higher learning, except where due acknowledgment has been made in the text. Duccio Migliorini 29/11/2015 A copy of the thesis will be available at http://www.dispaa.unifi.it/ Dichiarazione Con la presente affermo che questa tesi è frutto del mio lavoro e che, per quanto io ne sia a conoscenza, non contiene materiale precedentemente pubblicato o scritto da un'altra persona né materiale che è stato utilizzato per l’ottenimento di qualunque altro titolo o diploma dell'Università o altro istituto di apprendimento, a eccezione del caso in cui ciò venga riconosciuto nel testo. Duccio Migliorini 29/11/2015 Una copia della tesi sarà disponibile presso DISPAA , sez. Patologia Vegetale ed Entomologia, http://www.dispaa.unifi.it/ 2 RIASSUNTO Parole chiave: qPCR; Phytophthora; invasive micro-organism; disease management; early detection, aerial spread, soilborn pathogen, Janzen-Connell negative density dependence hypothesis Scopi Scopo della tesi è stato l’analisi della patogenicità ed ecologia del genere Phytophthora attraverso differenti approcci molecolari. Metodi e Risultati L’efficacia di PCR quantitativa basata su una sonda TaqMan specifica per il genere è stata confermata come preciso strumento di rilevamento di DNA di Phytophthora su suolo artificialmente infettato, in suolo di invasatura contaminato proveniente da vivaio ed in campioni di trappole aeree. Nessuna quantificazione di DNA è avvenuta dopo due settimane dalla morte indotta del patogeno e, in confronto con i metodi di isolamento tradizionali, è stata dimostrata una significativa maggiore efficienza come strumento diagnostico. La fluttuazione stagionale di Phytophthora in aria è stata quantificata e descritta per il periodo di campionamento. La tecnica di sequenziamento 454 è stata utilizzata per identificare la tassonomia delle specie di Phytophthora in un hotspot biologico in Western Australia ed al fine di descrivere la patogenicità delle due specie sequenziate con maggior frequenza è stato realizzato un esperimento in serra. Conclusioni Le tecniche di laboratorio utilizzate in questo studio hanno fornito nuove nozioni sull’ecologia di Phytophthora. La PCR quantitativa basata su sonda TaqMan è testata e proposta come efficace strumento di prevenzione verso l’arrivo di specie invasive. Importanza dell’impatto di questo studio L’efficienza della gestione di Phytophthora dipende dalla conoscenza delle caratteristiche patogene di gruppi ristretti di specie o di una singola specie . Ricerche come queste forniscono le basi scientifiche per comprendere l’epidemiologia di una malattia ed applicare un controllo risolutivo. Lavori correlati alla Tesi. Migliorini, D., Ghelardini, L., Tondini, E., Luchi, N., & Santini, A., 2015, The potential of symptomless potted plants for carrying invasive soilborne plant pathogens, Diversity and Distributions, 21(10), 1218-1229.* * Articolo riprodotto con il permesso di Diversity and Distributions 3 ABSTRACT Key words qPCR; Phytophthora; invasive micro-organism; disease management; early detection, aerial spread, soil born pathogen, Janzen-Connell negative density dependence hypothesis Aims The purpose of my thesis was to investigate pathogenicity and ecological traits of the genus Phytophthora through molecular approaches. Methods and Results A quantitative PCR technique based on a genus specific TaqMan probe was confirmed as a precise method for detecting Phytophthora DNA in artificially infested soil under laboratory condition, in naturally infested soil and tissues of potted nursery plants and in the filters of air traps. No positive DNA quantification occurred in soil after two weeks from pathogen induced death and a significant higher efficiency as diagnostic tool was demonstrated compared to traditional isolation methods both in soil and plant tissues. Seasonal fluctuation of aerial spread of Phytophthora was also quantified and described. A 454 sequencing approach was used to identify the Phytophthora species present in a biological hotspot area in Western Australia, and a glass house experiment was performed in order to describe the pathogenicity traits of the two most frequently detected species. Conclusions The lab procedures used in this study provided a more precise knowledge of Phytophthora ecology. The quantitative PCR assay based on designed TaqMan probe was demonstrated to be very efficient and is proposed as a reliable early detection instrument of prevention against the income of invasive species. Significance of impact of the study Efficient management of Phytophthora depends to the knowledge of pathogenicity traits in restrict groups or single species. Investigations like those presented in this thesis contribute the scientific bases to understand the epidemiology of disease and to apply a successful control. Papers related to the Thesis. Migliorini, D., Ghelardini, L., Tondini, E., Luchi, N., & Santini, A., 2015, The potential of symptomless potted plants for carrying invasive soilborne plant pathogens, Diversity and Distributions, 21(10), 1218-1229.* * Article reproduced with permission of Diversity and Distributions 4 Contents Introduction................................................................................................................................................... 6 Biological cycle of Phytophthora................................................................................................................ 6 The study of Phytophthora as a plant pathogen ....................................................................................... 7 Aim............................................................................................................................................................... 10 Materials and methods................................................................................................................................ 11 I The potential of symptomless potted plants for carrying invasive soil-borne plant pathogens............ 11 II A reliable lab procedure to detect the presence of viable Phytophthora inoculum in soil samples through qPCR........................................................................................................................................... 12 III Real Time PCR detection of airborne Phytophthora species in a plant nursery area.......................... 13 IV Phytophthora distribution, taxonomy and pathogenicity within a hyperdiverse bush plant hotspot in a dune system of Western Australia........................................................................................................ 14 Results.......................................................................................................................................................... 15 I The potential of symptomless potted plants for carrying invasive soil-borne plant pathogens............ 15 II A reliable lab procedure to detect the presence of viable Phytophthora inoculum in soil samples through qPCR........................................................................................................................................... 15 III Real Time PCR detection of airborne Phytophthora species in a plant nursery area.......................... 16 IV Phytophthora distribution, taxonomy and pathogenicity within a hyperdiverse bush plant hotspot in a dune system of Western Australia........................................................................................................ 16 Discussions................................................................................................................................................... 17 Bibliography................................................................................................................................................. 18 5 Introduction Biological cycle of Phytophthora The genus Phytophthora includes fungal-like microorganisms belonging to the Oomycetes (Peronosporaceae, Peronosporales, Peronosporidae, Peronosporea, Incertae sedis, Oomycota, Chromista). These microorganisms differ from fungi in as much as they produce zygotes, from which a diploid mycelium develops, and have cell walls composed of cellulose and glucans (Agrios, 2005). Figure 1. http://forestphytophthoras.org/. Sexual reproductive long term survival spores (Oospores) are generated when the antheridium