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Khdiar2018.Pdf Detection, Assessment, and Management of Phytophthora species in an Urban Forest by Mohammed Y. Khdiar (MSc) This thesis is submitted for the degree of Doctor of Philosophy School of Biological Sciences and Biotechnology Murdoch University Perth, Western Australia 2018 I Declaration I declare that this thesis is my own account of my research and contains as its main content work which has not previously been submitted for a degree at any tertiary education institution Mohammed Y. Khdiar July 2018 II Abstract Urban forests contribute to human well-being and environmental health and function, and this role will increase as urbanization continues to grow. Due to anthropogenic activities, urban forests are considered a suitable starting point for invasive pathogens. Members of the genus Phytophthora are important pathogens involved in tree and forest declines worldwide. To date, there are approximately 160 formally described Phytophthora species, with 50% of these species described in the last decade. There are many factors contributing to these recent descriptions, including introductions from the nursery trade, increased studies in forests and natural environments, and an increase in the number of Phytophthora scientists globally. Improved molecular tools to detect and identify Phytophthora species (as like other genera of the oomycetes) has also had a profound effect on the increased number of species that have recently been described. Within urban forests, trees are under considerable stress from polluting agents, mechanical damage, and other human activities and are often in sub-optimum health and are thus more vulnerable to pathogens such as Phytophthora. Two hundred and thirty-six soil and root sites were sampled from declining native Australian trees in 91 parks and nature reserves in the City of Joondalup, Perth Western Australia. Metabarcoding DNA detected forty-five Phytophthora species. Phytophthora multivora was isolated the most frequently while P. cinnamomi was the fifth most frequent. Seven species P. capsici, P. sp. pecan, P. fluvialis, P. gonapodyides, P. sp. walnut, P. erythroseptica and P. fallax were isolated only once. In contrast, only P. nicotianae, P. multivora, P. boodjera and P. arenaria were isolated into the culture based on rhizosphere baiting. A range of environmental factors was examined to determine if Phytophthora communities were related to specific environmental factors. The effect of environmental factors ranged from no effect (land class, plant diversity, park type, soil type, soil unit and canopy health) to a slight effect (canopy cover and park size) on the overall Phytophthora community. When looking at the incidence of P. cinnamomi, park type was significant, while for P. multivora, soil type, soil unit, and park size were significant drivers. III A pathogenicity test was conducted on excised branches from 15 tree species underbark inoculated with 19 Phytophthora species. All Phytophthora species formed lesions in Eucalyptus marginata and Corymbia calophylla, while Fraxinus excelsior was resistant to 7 of the 19 Phytophthora species. Six Phytophthora species were pathogenic to all 15-tree species, whereas Phytophthora versiformis formed lesions in only three tree species. Phytophthora cinnamomi, P. pseudocryptogea, and P. citrophthora were the most pathogenic species causing the largest lesions in most of these tree species. These results show that Phytophthora species potentially play an essential role in the declining health of urban trees and indicate the importance of managing Phytophthora in urban forests. Two chelate complexes [Zn (Val)2(bipy)] and [Ca(Val)2(bipy)] were developed and screened for their potential as fungicides to control Phytophthora species. These complexes were examined using three spectroscopic methods (X-ray crystallography, ultraviolet rays–visible spectra and Fourier-transform infrared spectroscopy). The complexes were pseudo-octahedral in structure, and both complexes had the same patterns of crystal structure symmetry. The yield of the calcium chelate and zinc chelate of basic materials were 81% and 50%, respectively. The activity of these complexes with or without the addition of phosphite against P. cinnamomi in vitro and in planta was examined. In vitro, the effect of the chemical treatments was assessed by measuring the EC50 of mycelial biomass in a liquid medium with 0-160 µg/ml (0-0.16g/l) of each complex. Phytophthora cinnamomi was highly sensitive to the Ca chelate alone and Ca chelate + phosphite, but not to the zinc chelate. Two plants species Banksia grandis and Eucalyptus marginata were sprayed with three concentrations (0, 0.25 and 0.5%) of each chemical treatment (phosphite, Ca chelate, Zn chelate and Ca chelate + phosphite) and were inoculated with P. cinnamomi together with a non-inoculated control. Ca chelate significantly reduced lesion development of P. cinnamomi compared to phosphite alone. Whilst Ca chelate (0,25%) + phosphite (0,25%) gave the highest improvement in reducing the effects of P. cinnamomi in both plant species. None of the Zn chelate applications were effective. The Ca chelate has the potential to be used as a fungicide to control Phytophthora species. These results suggest that Ca chelate may be a suitable alternative to phosphite to control Phytophthora species. IV Table of contents 1 Chapter One ................................................................................................................... 1 Literature Review .................................................................................................................. 1 1.1 General Introduction ............................................................................................... 2 1.2 Why are urban forests important? ........................................................................... 3 1.3 Tree Decline Overview ............................................................................................. 4 1.4 The role of Phytophthora in tree declines ................................................................. 5 1.5 The effect of predisposing and inciting factors on trees decline caused by Phytophthora species ......................................................................................................... 6 Predisposing factors ...................................................................................................................6 Inciting factors ............................................................................................................................9 1.6 New technologies to study urban tree health ......................................................... 11 High-throughput sequencing ..................................................................................................... 11 Remote sensing ........................................................................................................................ 12 1.7 Management of Phytophthora species with chemical applications ......................... 13 1.8 Status of knowledge of Phytophthora species associated with urban forests in Perth, Western Australia ............................................................................................................ 15 Perth, Western Australia ........................................................................................................... 15 Urban forests as an initial point of spread for Phytophthora species .......................................... 15 1.9 Summary ............................................................................................................... 16 1.10 Thesis Aims ............................................................................................................ 17 2 Chapter Two ................................................................................................................. 18 Association of Phytophthora with declining vegetation in an urban forest environment ....... 18 2.1 Abstract ................................................................................................................. 19 2.2 Introduction .......................................................................................................... 20 2.3 Materials and Methods .......................................................................................... 22 Study area and sample collection .............................................................................................. 22 Baiting technique ..................................................................................................................... 24 eDNA extraction from fine roots and metabarcoding ................................................................. 24 Accompanying data .................................................................................................................. 25 Data analysis ............................................................................................................................ 26 2.4 Results ................................................................................................................... 27 V Isolation by baiting ................................................................................................................... 27 Phytophthora species detected from fine root eDNA ................................................................. 27 Phytophthora community ........................................................................................................
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