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New Phytophthora Species in Western Australia: Pathogenicity and Control by Phosphite in Vitro and in Planta

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New Phytophthora Species in Western Australia: Pathogenicity and Control by Phosphite in Vitro and in Planta New Phytophthora species in Western Australia: pathogenicity and control by phosphite in vitro and in planta Rajah Belhaj B.Sc. Botany (Elmergib University, Libya) MSc Plant Physiology (Elmergib University, Libya) Thesis submitted for the degree of Doctor of Philosophy School of Veterinary and Life Sciences Murdoch University October 2017 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. To the best of my knowledge, all work performed by others, published or unpublished, has been acknowledged. Rajah Belhaj October 2017 ii Thesis abstract A number of new Phytophthora species to Western Australia, have been isolated from dying native species in southwest Western Australia (SWWA). However, little is known about their host range or whether the pathogenic species can be controlled by phosphite. The effect of phosphite on 23 Phytophthora species was investigated in vitro. On solid medium, EC50 values were 5 to >160 µg/ml phosphite, whilst in liquid medium buffered using 0.03 M MES (2-(N-morpholino) ethanesulfonic acid), EC50 values ranged from 30 to >900 µg/ml. Nineteen of 23 species displayed a much higher level of tolerance in buffered liquid medium to phosphite than in solid, or unbuffered liquid medium. Assessment of phosphite tolerance was more accurate using liquid, rather than solid medium. Casuarina obesa, Banksia littoralis, B. occidentalis, B. grandis, Lambetia inermis, Corymbia calophylla, and Eucalyptus marginata were screened in the glasshouse as possible susceptible hosts by inoculating soil with 22 Phytophthora species and assessing plant growth after 6 weeks. P. niederhauserii had a wide host range similar to P. cinnamomi. Other species that killed one or more hosts were P. elongata, P. boodjera, P. moyootj, P. constricta and P. rosacearum. No Phytophthora species tested killed C. calophylla. To test the effectiveness of phosphite as a control agent, Eucalyptus marginata, B. occidentalis, B. littoralis and L. inermis were sprayed with 0.5% phosphite seven days before soil inoculation with ten Phytophthora species. No phosphite-treated plants died, but in unprotected controls P. cinnamomi and P. neiderhauserii killed at least one host plant of all species, while plants of E. marginata were also killed by P. iii constricta, P. boodjera, P. elongata, and P. multivora. For P. constricta, P. boodjera, P. multivora, P. rosacearum and P. arenaria, for one or more host species, the reduction of shoot or root growth caused by the pathogen was not eliminated by spraying plants with phosphite. There was no relationship between phosphite tolerance in vitro (as determined by EC50) and the response to phosphite in planta: for example, P. gibbosa was highly tolerant in vitro but controlled by phosphite in planta. In L. inermis subsp. inermis all species of Phytophthora reduced root mass even in plants sprayed with phosphite. Phosphite prevented lesion development in E. marginata and B. occidentalis after underbark inoculation of with Phytophthora cinnamomi and P. niederhauserii. iv Table of Contents Declaration ............................................................................................................ ii Thesis abstract .................................................................................................... iii List of Publications ............................................................................................ vii Statement of Contributions.............................................................................. viii Acknowledgements ............................................................................................ ix CHAPTER 1 ........................................................................................................... 1 Literature review .......................................................................................................... 1 Phytophthora diseases ...................................................................................................... 2 Phytophthora cinnamomi ................................................................................................... 3 Discovery, origin and distribution ............................................................................................... 4 Occurrence in Australia ............................................................................................................... 4 Host range ..................................................................................................................................... 5 Infection process and disease expression ................................................................................ 7 Reproduction and survival ........................................................................................................... 8 The effect of phosphite on Phytophthora ................................................................................ 10 Uptake of phosphite and its translocation in plants ............................................................... 13 Phosphite phytotoxicity .............................................................................................................. 14 New species of Phytophthora in Australia .................................................................... 15 CHAPTER 2 ......................................................................................................... 22 In vitro growth of some Phytophthora species in solid and liquid media amended with phosphite .......................................................................................... 22 Abstract .............................................................................................................................. 23 Introduction ........................................................................................................................ 23 Materials and Methods .................................................................................................... 27 Phytophthora species tested for sensitivity to phosphite. ..................................................... 27 Preparation of solid and liquid media with phosphite ............................................................ 28 Experiment 1. The effect of phosphite on some Phytophthora species grown on solid RMM. ............................................................................................................................................ 30 Experiment 2. Preliminary experiments to determine the methods for assessment of growth in liquid RMM and to make comparisons with solid RMM. ...................................... 31 Experiment 4. Use of phosphite as a phosphate source by Phytophthora. ....................... 33 Results ............................................................................................................................... 34 Experiment 1. The effect of phosphite on some Phytophthora species grown on solid medium. ....................................................................................................................................... 34 Experiment 3. The effect of phosphite on some species of Phytophthora in vitro in liquid RMM amended with 0.03 M MES. ........................................................................................... 42 Experiment 4. Use of phosphite as a phosphate source by Phytophthora. ................................ 44 CHAPTER 3 ......................................................................................................... 48 Pathogenicity of 21 newly described Phytophythora species against seven WA native plants ............................................................................................................... 48 Abstract .............................................................................................................................. 49 Introduction ........................................................................................................................ 50 v Materials and methods .................................................................................................... 52 Host plant species ...................................................................................................................... 52 Phytophthora isolates and inoculum preparation ................................................................... 53 Growth of plants and inoculation .............................................................................................. 55 Assessment of host plants at harvest and re-isolation of Phytophthora ............................ 56 Data analysis ............................................................................................................................... 59 Eucalyptus marginata................................................................................................................. 60 Corymbia calophylla ...................................................................................................................... 60 Bankisia occidentalis .................................................................................................................. 61 Banksia littoralis ............................................................................................................................ 62 Banksia grandis .........................................................................................................................
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