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New Tools for the Detection of Phytophthora Cinnamomi in Environmental Samples

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New Tools for the Detection of Phytophthora Cinnamomi in Environmental Samples New tools for the detection of Phytophthora cinnamomi in environmental samples By Manisha B Kunadiya BSc Microbiology (Gujarat University), MSc Microbiology (Saurashtra University) Thesis submitted for the degree of Doctor of Philosophy School of Veterinary and Life Sciences Murdoch University December 2018 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. ……………………………………… Manisha B Kunadiya 21 December 2018 i Abstract Phytophthora cinnamomi (Rands) is one of the world’s most invasive plant pathogens and accurate diagnosis of its presence in plants and soil using molecular tools is very important. Few of the existing primers were found to discriminate between P. cinnamomi and a number of newly described species, and for those that could do so, the sensitivity was inadequate. Further, for my research, primers to detect both DNA and RNA were required, and the existing primers based on non-protein coding gene regions were inappropriate. New primers were developed based on the cytochrome oxidase subunit 2 (cox2) gene, a mitochondrial gene without introns and suitable for the RT-qPCR assay and applicable to both DNA and RNA. Procedures were modified to minimize loss of nucleic acids during extraction. These primers were specific for P. cinnamomi and able to detect as little as 150ag DNA. An exception was the closely related P. parvispora, which showed late amplification at high DNA concentrations. Primers were successfully used to detect infection in plant materials and in a range of soil types. The rate of decay of P. cinnamomi DNA and RNA in different soil types, under wet or dry conditions were also studied. P. cinnamomi DNA can survive in soil with no living host plant roots, for 378 days or more if the soil is dry, but only up to 90 days if it is wet. P. cinnamomi RNA can persist in soil for only 3 days or less in both dry and wet soil; in wet silty loam it could not be recovered after ~30 minutes. The clay content of the soil also affected the survival time of the DNA. Although RNA analysis is very accurate for the detection of living P. cinnamomi, the high cost of the analysis makes it impractical for widespread use at present. The new primers have already been adopted by the Centre for Phytophthora Science and Management as part of a best-practice protocol used to determine is P. cinnamomi is still present following eradication activities on Alcoa mine sites. ii Table of Contents DECLARATION ................................................................................................................................................... I ABSTRACT ........................................................................................................................................................ II ACKNOWLEDGEMENT ..................................................................................................................................... V LIST OF PUBLICATION .................................................................................................................................... VI JOURNAL ARTICLES ................................................................................................................................................. VI CONFERENCE PRESENTATION .................................................................................................................................... VI CHAPTER 1: LITERATURE REVIEW ..................................................................................................................... 1 INTRODUCTION ....................................................................................................................................................... 2 DISEASE CYCLE OF PHYTOPHTHORA ............................................................................................................................. 6 CONTROL OF PHYTOPHTHORA SPECIES ...................................................................................................................... 10 ISOLATION AND IDENTIFICATION OF PHYTOPHTHORA SPECIES ......................................................................................... 12 GENE REGIONS USED IN THE IDENTIFICATION OF PHYTOPHTHORA SPECIES......................................................................... 14 MOLECULAR DIAGNOSTICS ..................................................................................................................................... 17 THESIS AIMS AND RESEARCH OBJECTIVES: ................................................................................................................... 24 CHAPTER 2: PATHWAYS TO FALSE POSITIVE DIAGNOSES USING MOLECULAR GENETIC DETECTION METHODS; PHYTOPHTHORA CINNAMOMI AS A CASE STUDY. ......................................................................................... 26 ABSTRACT ........................................................................................................................................................... 27 INTRODUCTION ..................................................................................................................................................... 28 MATERIALS AND METHODS .................................................................................................................................... 30 RESULTS AND DISCUSSION ...................................................................................................................................... 34 CONCLUSION ....................................................................................................................................................... 36 CHAPTER 3: A QPCR ASSAY FOR THE DETECTION OF PHYTOPHTHORA CINNAMOMI INCLUDING AN MRNA PROTOCOL DESIGNED TO ESTABLISH PROPAGULE VIABILITY IN ENVIRONMENTAL SAMPLES ........................ 38 ABSTRACT ........................................................................................................................................................... 39 INTRODUCTION ..................................................................................................................................................... 40 MATERIALS AND METHODS .................................................................................................................................... 42 RESULTS .............................................................................................................................................................. 48 COMPARISON OF RNA EXTRACTION KITS ................................................................................................................... 56 DETECTION OF P. CINNAMOMI IN ASYMPTOMATIC BAITS ............................................................................................... 57 DISCUSSION ......................................................................................................................................................... 58 CHAPTER 4: DIFFERENT SOIL TYPES VARY CONSIDERABLY IN THEIR INHERENT MICROBIAL POTENTIAL TO DEGRADE NUCLEIC ACIDS (DNA AND RNA) .................................................................................................... 61 iii ABSTRACT ........................................................................................................................................................... 62 INTRODUCTION ..................................................................................................................................................... 63 MATERIALS AND METHODS .................................................................................................................................... 66 RESULTS .............................................................................................................................................................. 70 CHARACTERISATION OF THE SOIL PHYSICAL AND CHEMICAL PROPERTIES ............................................................................ 70 DISCUSSION ......................................................................................................................................................... 78 CHAPTER 5: BEST PRACTICE PROTOCOL FOR THE MOLECULAR DETECTION OF P. CINNAMOMI IN AUSTRALIA ...................................................................................................................................................................... 83 INTRODUCTION ................................................................................................................................................ 84 TAXONOMIC INFORMATION ............................................................................................................................ 85 DETECTION ....................................................................................................................................................... 85 DETECTION IN PLANT MATERIAL ............................................................................................................................... 91 IDENTIFICATION ............................................................................................................................................... 94 CHAPTER 6: GENERAL DISCUSSION .............................................................................................................. 100 MAJOR FINDINGS OF THE PROJECT .........................................................................................................................
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