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Understanding Pasture Performance: the Interaction Between Perennial Ryegrass and Viruses

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Understanding Pasture Performance: the Interaction Between Perennial Ryegrass and Viruses Lincoln University Digital Thesis Copyright Statement The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). This thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: you will use the copy only for the purposes of research or private study you will recognise the author's right to be identified as the author of the thesis and due acknowledgement will be made to the author where appropriate you will obtain the author's permission before publishing any material from the thesis. Understanding Pasture Performance - The Interaction between Perennial Ryegrass and Viruses A thesis submitted in partial fulfilment of the requirements for the degree of Master of Science at Lincoln University Maxine Farquhar 2016 Abstract In New Zealand livestock-based agriculture is an essential component of the economy, and at the very core of the majority of livestock-based farming systems in New Zealand is a pasture comprised of perennial ryegrass (Lolium perenne L.). Therefore, the productivity and profitability of livestock industries is inextricably linked to, and influenced by, the quality and performance of the underlying perennial ryegrass pasture. However, previous research indicates that ryegrass pastures in New Zealand could be infected with multiple viruses, and that the incidence or “load” of viruses within ryegrass pastures could potentially increase over time. Considering the significance of ryegrass to agricultural productivity, and current targets to increase the output of ryegrass pastures, these findings are concerning, as 1) multiple virus infections could have synergistic effects, in which the impact of one pathogen could potentially be exacerbated by the concurrent presence of other pathogens within a single host, and 2) increasing viral load or incidence within a pasture could potentially undermine the productivity and persistence of a ryegrass pasture over time. However, the extent to which multiple virus infections and/or increasing viral load undermines the persistence and productivity of perennial ryegrass is yet to be ascertained. Therefore, the purpose of this research is to elucidate the potential impact of multiple virus infections and increasing viral load upon the yield and persistence of ryegrass. To determine the impact of viral load and/or multiple viruses upon ryegrass, tillers obtained from 10-year-old and 1-year-old ryegrass were screened for multiple viruses, viral load was quantified, and the yield of old and new ryegrass was compared over time, to ascertain if there was or is potentially a link between viral load and ryegrass performance. The results of this project indicate 1) that multiple viruses are present within the ryegrass material examined, 2) that viral load is higher in 10-year-old ryegrass, and 3) 10-year-old old ryegrass produced less biomass (4-29%) than young ryegrass. Overall, the results of this research demonstrate that there is a potential correlation or link between increasing and/or high viral load and yield deficit in old ryegrass. 2 Table of contents Abstract .................................................................................................................................... 2 Table of contents ..................................................................................................................... 3 List of Figures .......................................................................................................................... 5 List of Tables… ....................................................................................................................... 6 Abbreviations… ...................................................................................................................... 7 Chapter One: Introduction .................................................................................................... 8 1.1 Ryegrass - Traits of Priority ................................................................................... 8 1.2 What are Viruses? .................................................................................................. 9 1.2.1 Plant Viruses – The Global Context ...................................................................... 9 1.3 Plant Viruses and New Zealand Ryegras .............................................................. 10 1.3.1 Barley Yellow Dwarf Virus ................................................................................... 11 1.3.2 Ryegrass Mosaic Virus.......................................................................................... 20 1.3.3 Lolium perenne Partitivirus .................................................................................. 26 1.3.4 Multiple Virus Infection & Ryegrass .................................................................... 30 1.4 Other Implications of Plant Viruses ...................................................................... 30 1.5 Research Objectives .............................................................................................. 32 Chapter Two: Materials and Methods ................................................................................. 34 Technical Objective #1 – Comparative analysis of ryegrass yield and persistence .............. 34 2.1 Biological material ............................................................................................... 35 2.2 Development of the field trial site......................................................................... 37 2.3 Standardized sampling and storage protocol ........................................................ 40 Technical Objective #2 – Detection and quantification of viruses ........................................ 41 2.4 Sample acquisition and handling .......................................................................... 43 2.5 RNA isolation and reverse transcription ............................................................... 44 2.6 Primer Design ....................................................................................................... 47 2.6.1 Primers for BYDV ................................................................................................ 47 2.6.2 Primers for RGMV................................................................................................ 49 2.6.3 Primers for LPPV .................................................................................................. 50 3 2.6.4 Validation of Primer.............................................................................................. 50 2.7 qRT-PCR ............................................................................................................... 51 2.7.1 Establishment of a Standard Curve ....................................................................... 51 2.7.2 qRT-PCR assays ................................................................................................... 53 2.7.3 Replicates and Controls ........................................................................................ 53 2.7.4 Converting Cq Values to Copy Number ............................................................... 55 2.7.5 Normalisation of qPCR data ................................................................................. 56 2.7.6 Statistical Analysis of qRT-PCR data ................................................................... 57 2.8 ELISA ................................................................................................................... 58 2.9 siRNA.................................................................................................................... 58 Chapter Three: Results ......................................................................................................... 59 Results – Technical Objective #1 .......................................................................................... 59 3.1 Field Site & Turf Grass Yield ............................................................................... 59 3.2 Ryegrass Yield ...................................................................................................... 59 3.3 Ryegrass Persistence ............................................................................................. 62 Results – Technical Objective #2 .......................................................................................... 65 3.4 ELISA results ........................................................................................................ 65 3.5 siRNA results ........................................................................................................ 66 3.6 qRT-PCR results ................................................................................................... 68 3.6.1 BYDV ................................................................................................................... 68 3.6.2 RGMV ................................................................................................................... 74 3.7 Results Summary .................................................................................................. 78 Chapter Four: Discussion 4.1 Ryegrass & Viral Load ........................................................................................ 79 4.2 Ryegrass Yield and Persistence ............................................................................ 81 4.3 Co-infection of Ryegrass .....................................................................................
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