LIGHT LEAF SPOT AND WHITE LEAF SPOT OF BRASSICACEAE IN WASHINGTON STATE By SHANNON MARIE CARMODY A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN PLANT PATHOLOGY WASHINGTON STATE UNIVERSITY Department of Plant Pathology JULY 2017 © Copyright by SHANNON MARIE CARMODY, 2017 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the thesis of SHANNON MARIE CARMODY find it satisfactory and recommend that it be accepted. Lindsey J. du Toit, Ph.D., Chair Lori M. Carris, Ph.D. Timothy C. Paulitz, Ph.D. Cynthia M. Ocamb, Ph.D. ii ACKNOWLEDGMENT I would like to thank my major advisor Dr. Lindsey du Toit for her tireless mentorship, passion, and enthusiasm. I wish to thanks my committee members Dr. Lori Carris, Dr. Cynthia Ocamb, and Dr. Timothy Paulitz who welcomed me into their labs in Pullman, WA and when visiting in Corvallis, OR. This work would not have been possible without the financial support of the Clif Bar Family Foundation Seed Matters Initiative and the Western Sustainable Agriculture Research and Education Fellowship. Thank you to all of the faculty, students, and staff of WSU Mount Vernon and WSU Pullman who have generously shared time, support, knowledge, tulips, equipment, and humor. As was noted in my hospital chart, you all made sure I was “emotionally, financially, and botanically supported” which is more than I could have ever asked for. None of my research would have been possible without the members of the Vegetable Seed Pathology Lab. In particular, I’d like to thank Mike Derie for sharing his knowledge, Barbara Holmes who is just the best, and fellow students and future colleagues John Weber and Eric Christianson for being such a positive part of this experience. I would like to thank collaborators who contributed to this research – Tom Stearns from High Mowing Seed Co., Dr. Tobin Peever from WSU, and Drs. Kevin King and Jon West from Rothamsted Research – finding people to share ideas and discoveries with in the small world of seed pathology and P. brassicae research was so rewarding. This process was made meaningful by the many members of the Puget Sound Seed Growers Association who shared their farms, brassica seed, knowledge, and support. And to my husband Colin Curwen-McAdams, uva uvam vivendo varia fit, I love you. iii LIGHT LEAF SPOT AND WHITE LEAF SPOT OF BRASSICACEAE IN WASHINGTON STATE Abstract by Shannon Marie Carmody, M.S. Washington State University July 2017 Chair: Lindsey J. du Toit Pyrenopeziza brassicae, cause of light leaf spot of brassicas, was first found in the USA in 2014 in the Willamette Valley of Oregon. Neopseudocercosporella capsellae, cause of white leaf spot of brassicas, occurred rarely in the Pacific Northwest prior to being found across the Willamette Valley in 2014. In this study, a 2016 survey of northwestern Washington, a primary region of biennial brassica vegetable seed production for the USA, revealed both diseases to be present in mustard (Brassica juncea) cover crops and on bird’s rape mustard (B. rapa) weeds, but not in cabbage (B. oleracea var. capitata) seed crops. Sexual crossing tests, pathogenicity tests, and DNA phylogenetic analyses (latter of the internal transcribed spacer region of ribosomal DNA, β tubulin gene, translation elongation factor 1- gene, and mating type genes (MAT1-3 and MAT1-2); and multi-locus sequence analysis of the first three sequences) of P. brassicae isolates from the USA, European Union, New Zealand, and United Kingdom revealed that isolates from the USA likely represent a new species of Pyrenopeziza, hereafter referred to as P. cf. brassicae. P. cf. brassicae was demonstrated to be seedborne and seed transmitted on cabbage and mustard. Incubating infested seed on NP-10 agar medium at 4oC, followed by microscopic iv examination of the seed revealed P. cf. brassicae to be present on 12.50 to 19.75% of a mustard seed lot and <0.50% of a cabbage seed lot. Planting the infested mustard seed in a greenhouse resulted in a seed transmission rate of 0.1 to 5.3%. Seed treatment trials revealed that chlorine (1.2% NaOCl for 10, 20, 30, and 40 minutes), hot water (50oC for 15 and 30 minutes), steam (62.8, 65.6, 68.3, and 71.1oC for 90 seconds), and 10 fungicide seed treatments all reduced the incidence of mustard seed infected with P. cf. brassicae to <5%, and reduced seed transmission of the fungus from 3.4% for non-treated seed to <1%. Hot water and most of the steam treatments eradicated the pathogen from seed, but the hottest steam treatment was phytotoxic. The most efficacious fungicide seed treatments contained benzimidazole, a demethylation inhibitor, and/or strobilurin active ingredients. v TABLE OF CONTENTS Page ACKNOWLEDGMENTS ..................................................................................................................... iii ABSTRACT ........................................................................................................................................ iv LIST OF TABLES ............................................................................................................................... xii LIST OF FIGURES ............................................................................................................................ xiv CHAPTER ONE: LITERATURE REVIEW .............................................................................................. 1 1.1 Brassicaceae ................................................................................................................. 1 1.1.1 Brassica diversity: Brassica as a genus........................................................ 2 1.1.1.1 Brassica carinata A. Braun .............................................................. 3 1.1.1.2 Brassica juncea (L.) Czern & Coss.................................................... 3 1.1.1.3 Brassica napus L. ............................................................................. 4 1.1.1.4 Brassica nigra (L.) Koch ................................................................... 4 1.1.1.5 Brassica oleracea L. ......................................................................... 5 1.1.1.6 Brassica rapa L. ............................................................................... 5 1.1.1.7 Weedy Brassicaceae genera ........................................................... 6 1.1.2 Brassica production in the Pacific Northwest USA ..................................... 6 1.1.2.1 Fresh market and processing crops ................................................ 6 1.1.2.2 Cover crops ..................................................................................... 7 1.1.2.3 Seed crops ....................................................................................... 8 1.1.2.4 Oilseed and biofuel crops ............................................................... 8 1.1.3 Brassica diseases ......................................................................................... 9 vi 1.1.3.1 Brassica diseases in the Pacific Northwest USA ........................... 10 1.2 Light Leaf Spot [Cylindrosporium concentricum (Anamorph), Pyrenopeziza brassicae (Teleomorph)] ............................................................................................................ 14 1.2.1 Taxonomy .................................................................................................. 14 1.2.2 Morphology and physiology ..................................................................... 15 1.2.3 History, geographic distribution, and impact ........................................... 16 1.2.4 Host range ................................................................................................. 18 1.2.5 Symptoms ................................................................................................. 18 1.2.6 Disease cycle ............................................................................................. 19 1.2.6.1 Dispersal ........................................................................................ 19 1.2.6.2 Host plant infection ...................................................................... 20 1.2.6.3 Survival and seed transmission ..................................................... 21 1.2.7 Management ............................................................................................. 24 1.2.7.1 Cultural control ............................................................................. 24 1.2.7.2 Chemical control ........................................................................... 25 1.3 White Leaf Spot [Pseudocercosporella capsellae (Anamorph), Mycosphaerella capsellae (Teleomorph)] ............................................................................................ 27 1.3.1 Taxonomy .................................................................................................. 27 1.3.2 Morphology and physiology ..................................................................... 28 1.3.3 History, geographic distribution, and impact ........................................... 29 1.3.4 Host range ................................................................................................. 30 1.3.5 Symptoms ................................................................................................. 30 vii 1.3.6 Disease cycle ............................................................................................. 31 1.3.6.1 Dispersal .......................................................................................