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Of Phytophthora Cinnamomi (Rands) in Soil

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Of Phytophthora Cinnamomi (Rands) in Soil SURVIVAL OF DIFFERENT LIFE STAGES OF PHYTOPHTHORA CINNAMOMI (RANDS) IN SOIL AND PLANT ROOTS UNDER MINE SITE CONDITIONS By JAMBA GYELTSHEN B.Sc. Agri. (Kerela Agrl. Uni., India); M.Sc. Crop Protection (Uni. of Reading, U.K.); Doctor of Plant Medicine (Uni. of Florida, U.S.A.) A thesis presented to Murdoch University for the degree of Doctor of Philosophy May 2, 2018 DECLARATION I declare that this thesis is my own account of research, and contains as its main content, work which has not previously been submitted for a degree at any tertiary education institution. Jamba Gyeltshen ii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisors, Professor Giles Hardy, Dr. Treena Burgess, and Dr. Bill Dunstan for their profound inspiration, guidance, and support during the course of this study. Without that kind of support, it would not have been possible to complete this work on time. Their accessibility at all times and the wholehearted willingness to help was a humbling experience. Thanks are also due to Dr. Navid Moheimani (Supervisory Committee Chair) for his time and advice. I am particularly grateful for the whole-hearted support received from Diane White and Briony Williams in the laboratory, Dr. Mike Calver and Chris Shaw for statistical analysis, and all my graduate fellow-mates for various other support and cooperation. My special thanks to my wife, Kunzang Chhoden; daughters, Thinley Yangzom and Jamba Chhoden; and son, Tashi Tenzin, for their moral support, encouragement, and sacrifices. I also wish to thank Kuenga Nidup for the help rendered with photoshop to put together the pictures and graphs. Finally, I am greatly indebted to Murdoch University for the scholarship within the framework of the ARC-Linkage Project that enabled me to carry out this study. iii ABSTRACT Phytophthora cinnamomi (Rands) is a soil and water-borne plant pathogen, associated with a devastating dieback disease in the jarrah (Eucalyptus marginata) forest, in Western Australia. As the forest is an active site for extensive open-cut bauxite mining, it is a challenge to prevent the potential spread of P. cinnamomi. Despite years of research, we have not fully understood how P. cinnamomi survives the long, hot and dry Mediterranean summer in the jarrah forest ecosystem, particularly under mine site conditions where surface temperatures periodically reach 60 ºC. This study looked at the life span of oospores, chlamydospores and encysted zoospores under conditions typical of the mine sites. In particular, it compared the effect of dry and moist soil conditions on survival of oospores and chlamydospores. The study also examined the effects of exogenous materials (smoke water, fish emulsion, and the fungicides, ridomil and furalaxyl) that are known to have stimulatory or inhibitory effects on growth. These were shown not to have any significant impact on the survival structures. The findings indicate that oospores of P. cinnamomi survive in the soil for less than one year irrespective of soil moisture conditions, while chlamydospores survive for less than 12 weeks under similar conditions. Encysted zoospores under submerged conditions (similar to those in drainage sumps along haul roads) did not survive beyond one week. The effect of varying moisture levels was examined with oospores of a closely related species (P. multivora) which is associated with the dieback disease complex. At matric potentials between -1 kPa and -6 MPa, there was a clear decline in oospore viability, and at -6 MPa, which is the level of dryness typically experienced during the hot dry summers, oospore iv viability was reduced to 66% after 60 days. The possible role of non-susceptible or tolerant host plants growing on topsoil stockpiles on pathogen survival was investigated to determine how plants contribute to the long-term survival of the pathogen. Meta-barcoding and high throughput sequencing detected P. cinnamomi on 16 of the 20 plant species assessed. Unexpectedly, the technique revealed the putative presence of 24 other Phytophthora species, thereby raising more questions on the role of these plants in the Phytophthora disease cycle. Based on the findings, this study recommends that the stockpiles be maintained in situ and plant-free for at least 2-3 years to minimize the risk of spreading the pathogen and permanently removing the source of inoculum. v CONFERENCE PRESENTATION A presentation of the work (Chapter 2) was made at the 8th Congress of IUFRO Group 7.02.09: “Phytophthora in Forests and Natural Ecosystems” held in Sapa, Vietnam from 18th to 25th of March 2016. Title: The decline in viability of Phytophthora cinnamomi survival structures under moist and dry soil conditions. 1 1 1 1 Jamba Gyeltshen , William Dunstan , Treena Burgess , Giles Hardy 1Centre for Phytophthora Science and Management, School of Veterinary and Life Sciences, Murdoch University, W. Australia, 6150; [email protected] vi TABLE OF CONTENTS DECLARATION .............................................................................................................................. ii ACKNOWLEDGEMENTS ............................................................................................................ iii ABSTRACT ................................................................................................................................... iv CONFERENCE PRESENTATION ............................................................................................... vi CHAPTER 1: GENERAL INTRODUCTION AND LITERATURE REVIEW ..................................................... 1 1.1 GENERAL INTRODUCTION ................................................................................................. 1 1.1.1 Structure of the thesis .......................................................................................... 3 1.2 BIOLOGY AND LIFE CYCLE OF PHYTOPHTHORA CINNAMOMI ............................. 4 1.2.1 The phylogeny of Phytophthora cinnamomi ......................................................... 4 1.2.2 Morphology of Phytophthora cinnamomi .............................................................. 5 1.2.3 Reproduction and life cycle .................................................................................. 5 1.2.4 Dispersal ............................................................................................................. 8 1.2.5 Nutrition and the infection process ....................................................................... 8 1.2.6 Dormancy, viability, viability testing.................................................................... 10 1.2.7 Vital stain ........................................................................................................... 12 1.2.8 Survival of Phytophthora cinnamomi.................................................................. 14 vii 1.3 ECOLOGICAL FACTORS AFFECTING PHYTOPHTHORA CINNAMOMI .................... 22 1.3.1 Soil moisture (matric potential) .......................................................................... 23 1.3.2 Soil temperature ................................................................................................ 25 1.3.3 Microbial antagonism to Phytophthora ............................................................... 26 1.4 PHYTOPHTHORA DIEBACK AND BAUXITE MINING ........................................... 28 1.4.1 Jarrah forest environment & Phytophthora dieback............................................ 28 1.4.2 Host range of Phytophthora cinnamomi ............................................................. 29 1.4.3 Bauxite mining and rehabilitation ....................................................................... 30 1.4.4 Dieback management ........................................................................................ 31 1.5 THESIS AIMS AND RESEARCH OBJECTIVES ..................................................... 32 CHAPTER 2: HOW TIME, MOISTURE AND EXOGENOUS FACTORS AFFECT THE VIABILITY OF PHYTOPHTHORA CINNAMOMI PROPAGULES IN SOIL ............................................... 34 2.1 INTRODUCTION ..................................................................................................... 34 2.2 MATERIALS AND METHODS ........................................................................................ 37 2.2.1 Experiment 1: The effect of time, moisture level and exogenous factors on viability of oospores ........................................................................................... 39 2.2.2 Experiment 2: Determining the effect of time, moisture level and exogenous factors on viability of chlamydospores ................................................................ 44 2.2.3 Viability determination using Tetrazolium bromide stain (MTT) .......................... 45 2.2.4 Viability determination through RNA assay ........................................................ 48 2.2.5 Analysis of data ................................................................................................. 49 viii 2.3 RESULTS.......................................................................................................................... 50 2.3.1 Experiment 1: Determining the effect of time, moisture level and exogenous factors on viability of oospores ........................................................................... 50 2.3.2 Experiment 2: The effect of time, moisture level and exogenous factors on chlamydospore viability .....................................................................................
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