Suppression of the Root-lesion Nematode Using Liquid Hog Manure By AMRO MAHRAN A Thesis Submitted to the Faculty of Graduate Studies In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Plant Science University of Manitoba Winnipeg, Manitoba, Canada © Copyright by Amro Mahran 2009 THE UNIVERSITY OF MANITOBA FACULTY OF GRADUATE STUDIES ***** COPYRIGHT PERMISSION Suppression of the Root-lesion Nematode Using Liquid Hog Manure By AMRO MAHRAN A Thesis/Practicum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirement of the degree Of Doctor of Philosophy Amro Mahran © 2009 Permission has been granted to the Library of the University of Manitoba to lend or sell copies of this thesis/practicum, to the National Library of Canada to microfilm this thesis and to lend or sell copies of the film, and to University Microfilms Inc. to publish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made available by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws express written authorization from the copyright owner. ii ACKNOWLEDGMENTS I would like to express my deep and sincere appreciation to my advisor, Dr. Mario Tenuta, for his exceptional guidance, encouragement, support, and valuable suggestions he provided throughout the course of my program. I would like to express my sincere gratitude to my co-advisor, Dr. Fouad Daayf, for his constant encouragement, support, and guidance throughout my program. I sincerely thank Dr. Mark Hanson and Dr. Dilantha Fernando for serving in my advisory committee and for their valuable advice and effort during the course of my studies. I would like to thank Corinne Barker, Oscar Molina, Mervin Bilous, Lorne Adam, and Igor Lalin for technical assistance. I sincerely thank Dr. Manuel Mundo-Ocampo (Department of Nematology, University of California, Riverside) for production of the scanning electron micrographs. I would like to thank the Caenorhabditis Genetics Center, which is funded by the NIH National Centre for Research Resources, for supplying the C. elegans strain used in the bioassays and Dr. Tom Forge, Agriculture Canada, for supplying the Pratylenchus penetrans used in the bioassays. I sincerely thank the Agri-Food Research and Development Initiative, Ontario Pork, McCain Foods Ltd., Keystone Vegetable Producers Association, the J.R. Simplot Company, National Science and Engineering Research Council of Canada Discovery Grant Program (to Tenuta), and the Canada Research Chair Program for financial support. The financial support from the University of Manitoba Graduate Fellowship and the Government of Egypt is deeply acknowledged with gratitude. I owe my sincere loving thanks to my wife, Ola El-Maghraby, for her support and patience throughout the years of my studies. Without her love and understanding, this work would not have been completed. I thank my little princesses, Mariam and Tala, for their understanding as this thesis took a lot from their time. A special thank to my mother back in Egypt for her love, support, and prayers and for my brothers, Dr. Ali Mahran and Dr. Ahmed Mahran, for their backing and support. I would like to express my sincere thanks to my father-in-law, Dr. Mohamed El-Maghraby, for his continuous guidance and support. My appreciation to my mother-in-law for her loving support and prayers. I dedicate this thesis to my father. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ………………………………………………………….. iii LIST OF TABLES ………………………………………………………………........ viii LIST OF FIGURES ………………………………………………………………….. x LIST OF ABBREVIATIONS ………………………………………………………... xi ABSTRACT …………………………………………………………………………... xiii FORWARD …………………………………………………………………………… xv 1.0 INTRODUCTION ………………………………………………………………... 1 2.0 LITERATURE REVIEW………………………………………………………… 7 2.1 POTATO (SOLANUM TUBEROSUM L.), HOST PLANT………………………………. 7 2.1.1 History of the potato ………………………………………………………………………………. 9 2.1.2 Economical importance of potato ……………………………………………………………….. 10 2.1.3 Potato in Manitoba …………………………………………………………………………………. 11 2.1.4 Diseases of Potato ………………………………………………………………………………….. 12 2.2 ROOT-LESION NEMATODE (PRATYLENCHUS SPP.) ………………………………. 14 2.2.1 Taxonomy ………………………………………………………………………………………........ 14 2.2.2 Pratylenchus spp. life cycle ……………………………………………………………………….. 15 2.2.3 Pratylenchus spp. in Canada ……………………………………………………………………… 18 2.2.4 Pratylenchus spp. diagnosis ……………………………………………..…………………......... 22 2.2.4.1 Morphological identification …………………………………………………………… 22 2.2.4.2 Biochemical identification (Protein electrophoresis).……………………………….. 22 2.2.4.3 Molecular identification …………………………………………………………………. 25 2.2.5 Pratylenchus spp. pathogenicity ………………………………………………………………….. 28 2.2.5.1 Symptoms caused by Pratylenchus spp.………………………………………………… 28 2.2.5.2 Pratylenchus spp. host range …..………………………………………………….…... 29 2.2.5.3 Pratylenchus spp. damage thresholds ….……………………………………………….. 29 2.2.5.4 Pratylenchus spp. interaction with other plant pathogens …………………………… 30 2.2.6 Pratylenchus spp. management strategies ………………………………………………………. 36 2.2.6.1 Cultural practices ………………………………………………………………………… 37 iv 2.2.6.2 Physical control ……………………………………………………………………………. 47 2.2.6.3 Host plant resistance ……………………………………………………………………… 48 2.2.6.4 Biological control …………………………………………………………………………. 49 2.2.6.5 Chemical control ………………………………………………………………………….. 51 2.3 NEMATODE COMMUNITY STRUCTURE AS BIOINDICATOR OF SOIL HEALTH... 55 3.0 PREVALENCE AND SPECIES IDENTIFICATION OF PRATYLENCHUS SPP. IN MANITOBA POTATO FIELDS AND HOST SUITABILITY OF RUSSET BURBANK POTATO ….…………………………………………………. 58 3.1 ABSTRACT ……………………………………………………………………….. 58 3.2 INTRODUCTION ………………………………………………………………..... 59 3.3 MATERIALS AND METHODS ………………………………………………….. 61 3.3.1 Pratylenchus spp. prevalence in Manitoba………………………………………………………… 61 3.3.2 Morphological identification………………………………………………………………………… 62 3.3.3 Scanning electron microscope………………………………………………………………………. 63 3.3.4 Total DNA extraction…………………………………………………………………………………. 64 3.3.5 Amplification of the D3 region………………………………………………………………………. 65 3.3.6 Amplification using species-specific primers……………………………………………………… 65 3.3.7 DNA Sequencing………………………………………………………………………………………. 65 3.3.8 Basic Local Alignment Search (BLAST) …………………………………………………………... 66 3.3.9 Suitability of Russet Burbank potato as a host in commercial field conditions for a 66 population of Pratylenchus spp. in Manitoba……………………………………………………………. 3.3.10 Host suitability of Russet Burbank potato in growth chamber conditions to two 67 populations of Pratylenchus spp. in Manitoba…………………………………………………………… 3.3.11 Statistical analysis…………………………………………………………………………………… 69 3.4 RESULTS AND DISCUSSION …………………………………………............... 69 3.4.1 Pratylenchus spp. prevalence in Manitoba………………………………………………………… 69 3.4.2 Pratylenchus spp. identification…………………………………………………………………….. 70 3.4.3 Suitability of Russet Burbank………………………………………………………………………... 77 v 4.0 MORTALITY OF PRATYLENCHUS PENETRANS BY VOLATILE FATTY ACIDS FROM LIQUID HOG MANURE …………………..…………….. 82 4.1 ABSTRACT ……………………………………………………………………….. 82 4.2 INTRODUCTION………………………………………………………………...... 83 4.3 MATERIALS AND METHODS ………………………………………………….. 86 4.3.1 Manure collection and analysis…………………………………………………………………… 86 4.3.2 Nematode cultures……………………………………………………………………………………. 86 4.3.3 The bioassay…………………………………………………………………………………………… 88 4.3.4 Bioassay 1. Comparing toxicity of LHM and mixture of its VFA to P. penetrans……………. 89 4.3.5 Bioassay 2. Relative sensitivity of C. elegans and P. penetrans to VFA………………………. 89 4.3.6 Bioassay 3. Characterization of individual VFA lethality to P. penetrans……………………. 90 4.3.7 Bioassay 4. Interaction of individual VFA in the LHM in their toxicity to P. penetrans……. 90 4.3.8 Statistical analysis…………………………………………………………………………………….. 91 4.4 RESULTS …………………………………………………………………………. 92 4.4.1 LHM analysis………………………………………………………………………………………….. 92 4.4.2 Bioassay 1. Comparing LHM and a mixture of its VFA in their lethality to P. penetrans….. 92 4.4.3 Bioassay 2. Comparison of C. elegans and P. penetrans sensitivity to VFA………………….. 94 4.4.4 Bioassay 3. Screening of individual VFA based on their lethality to P. penetrans…………... 94 4.4.5 Bioassay 4. Interaction of individual VFA in the LHM in their lethality to P. penetrans…… 95 4.5 DISCUSSION……………………………………………………………………… 96 5.0 EFFECTIVENESS OF LIQUID HOG MANURE AND ACIDIFICATION TO KILL PRATYLENCHUS SPP. IN SOIL ………………………………………. 102 5.1 ABSTRACT ……………………………………………………………………….. 102 5.2 INTRODUCTION …………………………………………………………………. 103 5.3 MATERIALS AND METHODS ………………………………………………….. 106 5.3.1 Micro-plot Experiment 2004………………………………………………………………………… 106 5.3.2 Micro-plot Experiment 2005………………………………………………………………………… 110 5.3.3 Field Experiment……………………………………………………………………………………… 111 5.3.4 Nematode Analysis……………………………………………………………………………………. 113 5.3.5 pH and Volatile Fatty Acid Analysis………………………………………………………………. 113 vi 5.3.6 Statistical Analysis……………………………………………………………………………………. 114 5.4 RESULTS AND DISCUSSION …………………………………………………. 115 6.0 LIQUID HOG MANURE AND ITS ACIDIFICATION ALTERS A NEMATODE COMMUNITY IN SOIL …………………………………………….. 127 6.1 ABSTRACT ……………………………………………………………………….. 127 6.2 INTRODUCTION …………………………………………………………………. 128 6.3 MATERIALS AND METHODS ………………………………………………….. 131 6.3.1 Soil………………………………………………………………………………………………………. 131 6.3.2 Liquid Hog Manure…………………………………………………………………………………… 131 6.3.3