AN ASSESSMENT OF NEMATODES AFFECTING WHEAT IN MONTANA by Riyadh Talib Al-Khafaji A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Pathology MONTANA STATE UNIVERSITY Bozeman, Montana January 2018 ©COPYRIGHT by Riyadh Al-Khafaji 2018 All Rights Reserved ii DEDICATION To all scientists who are always searching to get truth of this universe. To my martyred father, Talib, in memoriam, and all freedom martyrs whose blood lit the path to liberty. To my mother Bedriah, my wife Ghusoon, my children Samaa, Ahmed, Talib, Tiba, Noor, and all my friends who have always given me unconditional advice and support. iii ACKNOWLEDGEMENTS I would like to thank and show my deep appreciation to my adviser, Dr. Alan T. Dyer, for guiding me patiently through my PhD program. I would like also express my gratitude to my committee members: Dr. Michael Giroux, Dr. William Grey, Dr. John Martin, and Dr. Kris Lambert, for their contributions to my success and knowledge growing through these years. I would also like to thank my lab members Erin Gunnink Troth, Jeff Johnston, Nathaniel Ellis, and David May for their tireless help and support. Thank you also to the members of the Department of Plant Science & Plant Pathology who have helped me throughout these studies. Thank you to Dr. Richard Smiley (Oregon State University) and Eileen Carpenter (Montana State University). Thank you to the Montana Extension agents who helped me do my survey across Montana. I am deeply grateful to the Republic of Iraq, especially the Establishment of Martyrs, for the scholarship and all the necessary support during my study. Thank you to the Ministry of Higher Education and Scientific Research, and the Iraqi Cultural office in Washington DC, for their help and support concerning formalities. Also, I would to thank the Montana Wheat and Barley Committee for funding these projects. iv TABLE OF CONTENTS 1. GENERAL INTRODUCTION .................................................................................. 1 Dissertation Organization ........................................................................................... 1 Cereal Crops............................................................................................................ 1 Montana Wheat and Barley .................................................................................... 2 The Phylum Nematoda and Plant Parasitic Nematodes .......................................... 2 Cereal Cyst Nematodes ........................................................................................... 6 Root Lesion Nematodes .......................................................................................... 9 The Root Lesion Nematode Pratylenchus spp. in Montana ................................. 11 Other Economically Significant Nematodes Identified in Montana ..................... 12 Thesis Justification ................................................................................................... 16 Literature Cited ......................................................................................................... 19 2. IDENTIFICATION OF PLANT PARASITIC NEMATODES ASSOCIATED WITH MONTANA WHEAT AND BARLEY .............................. 33 Abstract ..................................................................................................................... 33 Introduction .............................................................................................................. 34 Materials and Methods ............................................................................................. 36 Nematode Extraction from Soil Samples .............................................................. 38 Sieving and Sucrose Centrifugation.......................................................... 38 Sieving Method for Cyst Nematodes ........................................................ 39 Statistical Analysis of Soil and Cropping Systems ............................................... 39 Morphological Nematode Identification and Quantification ................................ 40 Molecular Nematode Identification ...................................................................... 40 Single Nematode DNA extraction ............................................................ 40 SSU rDNA Amplification ......................................................................... 41 Results ...................................................................................................................... 42 Pathogen Incidence ............................................................................................... 43 Effect of Soil and Cropping Systems. ................................................................... 45 Extraction Methods Correlate Significantly ......................................................... 48 Discussion ................................................................................................................. 49 Literature Cited ......................................................................................................... 59 v TABLE OF CONTENTS CONTINUED 3. DETERMINING VARIATION IN VIRULENCE AMONG POPULATIONS OF PRATYLENCHUS NEGLECTUS COLLECTED FROM MONTANA ......................................................................... 58 Abstract ..................................................................................................................... 58 Introduction .............................................................................................................. 59 Materials and Methods ............................................................................................. 62 Virulence of Pratylenchus neglectus on Montana crops ...................................... 63 Virulence of RLN on six sources of resistance in wheat ...................................... 64 Nematode extraction: Whitehead-Hemming method ........................................... 64 Morphological Measurements .............................................................................. 65 Single Nematode DNA Extraction ........................................................................ 66 Molecular Assessment .......................................................................................... 67 Statistical Analysis ................................................................................................ 68 Results ...................................................................................................................... 69 Variation in Virulence across Rotational Crops ................................................... 69 Nematode Population Response to Resistant Wheat Lines .................................. 69 Discussion ................................................................................................................. 71 Literature Cited ......................................................................................................... 82 REFERENCES CITED ................................................................................................ 87 vi LIST OF TABLES Table Page 1. Plant parasitic nematodes in Pacific Northwest on small grain crops which cause economic damage ..........................................................5 2. Other plant parasitic nematodes of widespread, but less serious, economic importance in the Pacific Northwest on small grains. ...........6 3. Putative species identification by classical morphological traits and by molecular barcoding and BLAST searches of standard nucleotide BLAST ...............................................55 4. Variation in reproduction factors across crops and RLN populations ...............74 5. Reproductive factors for different wheat lines across 8 populations of P neglectus for trial 1 ..................................................................75 6. Reproductive factors for different wheat lines, averaged across 8 populations of P neglectus for trial 2 ....................................76 7. Variation in physical characteristics among P. neglectus populations from different origins in Montana in 2017 at time point 1 (December 2016-March 2017) and time point 2 (March 2017-June 2017)...........................77 vii LIST OF FIGURES Figure Page 1. Map showing the Montana counties surveyed, in red, in both 2015 and 2016 .............................................................................53 2. Nematodes of significance in Montana. .............................................................54 3. Mean of root lesion nematodes Pratylenchus neglectus densities per kg dry soil by Whitehead trays in Montana counties in 2015 and 2016 ..................................................................................56 4. Mean cereal cyst nematode (Heterodera filipjevi and Heterodera avenae) densities per kg dry soil in Montana wheat and barley in 2015 and 2016, as extracted by the sieving method. ......................................57 5. Mean number of stunt nematodes (Tylenchorhynchus spp.) per kg dry soil in Montana wheat and barley in 2015 and 2016 as extracted by Whitehead trays. ........................................................................58 6. Percentage of Pratylenchus neglectus nematodes sampled from each population that were male, from wheat line experiment 1 ................78 7. Percentage of Pratylenchus neglectus nematodes sampled from each population that were male, from wheat line experiment 2 ................79 8.