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Project Title: Towards a Better Understanding of the Biology and Genetics of Phytophthora Rubi and Phytophthora Fragariae

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Project Title: Towards a Better Understanding of the Biology and Genetics of Phytophthora Rubi and Phytophthora Fragariae Project title: Towards a better understanding of the biology and genetics of Phytophthora rubi and Phytophthora fragariae Project number: CP 173 Project leader: Eleanor Gilroy, The James Hutton Institute (JHI), Dundee Report: Annual report, October 2019 Previous report: Annual report, October 2018 Key staff: Aurelia Bezanger (PhD student), Steve Whisson, Lydia Welsh, Ingo Hein Location of project: The James Hutton Institute, Dundee Industry Representative: Ross Mitchell, Castleton Fruit Ltd, Laurencekirk Date project commenced: October 2017 © Agriculture and Horticulture Development Board 2020. All rights reserved DISCLAIMER While the Agriculture and Horticulture Development Board seeks to ensure that the information contained within this document is accurate at the time of printing, no warranty is given in respect thereof and, to the maximum extent permitted by law the Agriculture and Horticulture Development Board accepts no liability for loss, damage or injury howsoever caused (including that caused by negligence) or suffered directly or indirectly in relation to information and opinions contained in or omitted from this document. © Agriculture and Horticulture Development Board 2018. No part of this publication may be reproduced in any material form (including by photocopy or storage in any medium by electronic mean) or any copy or adaptation stored, published or distributed (by physical, electronic or other means) without prior permission in writing of the Agriculture and Horticulture Development Board, other than by reproduction in an unmodified form for the sole purpose of use as an information resource when the Agriculture and Horticulture Development Board or AHDB Horticulture is clearly acknowledged as the source, or in accordance with the provisions of the Copyright, Designs and Patents Act 1988. All rights reserved. All other trademarks, logos and brand names contained in this publication are the trademarks of their respective holders. No rights are granted without the prior written permission of the relevant owners. The results and conclusions in this report are based on an investigation conducted over a one-year period. The conditions under which the experiments were carried out and the results have been reported in detail and with accuracy. However, because of the biological nature of the work it must be borne in mind that different circumstances and conditions could produce different results. Therefore, care must be taken with interpretation of the results, especially if they are used as the basis for commercial product recommendations. © Agriculture and Horticulture Development Board 2020. All rights reserved AUTHENTICATION We declare that this work was done under our supervision according to the procedures described herein and that the report represents a true and accurate record of the results obtained. Aurelia Bezanger PhD Student The James Hutton Institute, Dundee Signature ..... Date ............22/10/18................... Report authorised by: Dr Eleanor Gilroy Molecular Plant Pathologist The James Hutton Institute, Dundee Signature Date ...............................23/10/18............. © Agriculture and Horticulture Development Board 2020. All rights reserved CONTENTS GROWER SUMMARY .................................................................................................. 1 Headlines ..................................................................................................................... 1 Background and expected deliverables .................................................................. 1 Summary and the project’s main conclusions ....................................................... 1 Hydroponics raspberries ........................................................................................ 1 P. rubi isolation from canes ................................................................................... 4 P. rubi infection and life cycle ................................................................................ 5 Transgenic P. rubi ........................................................................................................................... 5 Sporangia and zoospores production ............................................................................................. 5 Infection in hydroponic conditions ................................................................................................. 5 P. rubi phenotypic diversity study ........................................................................ 6 Effects of the temperature ............................................................................................................. 6 Chemical screening......................................................................................................................... 7 P. rubi and P. fragariae genotypic diversity study: Pathogen Enrichment Sequencing (PenSeq) .............................................................................................. 9 Financial Benefits ..................................................................................................... 11 Action Points ............................................................................................................. 11 SCIENCE SECTION ................................................................................................... 13 Introduction ............................................................................................................... 13 Materials and methods ............................................................................................. 14 Hydroponics ............................................................................................................. 14 Raspberry cultures ........................................................................................................................ 14 Summary of hydroponics .............................................................................................................. 15 Cultures of Phytophthora rubi and Phytophthora fragariae .................................... 15 © Agriculture and Horticulture Development Board 2020. All rights reserved P. rubi infection and life cycle .................................................................................. 16 Transgenic P. rubi ......................................................................................................................... 17 Sporangia and zoospores production ........................................................................................... 18 Infection in hydroponic conditions ............................................................................................... 18 RNA extraction ............................................................................................................................. 19 Root staining ................................................................................................................................ 19 P. rubi phenotypic diversity study ...................................................................... 19 Effects of the temperature ........................................................................................................... 20 Fungicide screening ...................................................................................................................... 20 P. rubi and P. fragariae genotypic diversity study: Pathogen Enrichment Sequencing (PenSeq) ............................................................................................ 20 Baits library .................................................................................................................................. 21 Isolates collection ......................................................................................................................... 23 Target Enrichment Sequencing .................................................................................................... 23 Designing primers for qPCR to validate PenSeq ........................................................................... 23 Quantitative PCR set-up ............................................................................................................... 24 Results........................................................................................................................ 25 Hydroponics ............................................................................................................. 25 P. rubi infection and life cycle .................................................................................. 26 Life cycle of P. rubi ........................................................................................................................ 26 Root staining ................................................................................................................................ 28 P. rubi phenotypic diversity study ...................................................................... 28 Effects of the temperature ........................................................................................................... 28 Fungicide screening ...................................................................................................................... 29 P. rubi and P. fragariae genotypic diversity study: Pathogen Enrichment Sequencing (PenSeq) ............................................................................................ 32 qPCR ..........................................................................................................................................
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