Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2012 A Procedure Using Rotylenchulus Reniformis Nucleotide Sequences to Quantitatively Measure Plant-Parasitic Nematode Infestation Levels using Metagenomic Dna Isolated Directly from Soil Kurtis C. Showmaker Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Showmaker, Kurtis C., "A Procedure Using Rotylenchulus Reniformis Nucleotide Sequences to Quantitatively Measure Plant-Parasitic Nematode Infestation Levels using Metagenomic Dna Isolated Directly from Soil" (2012). Theses and Dissertations. 233. https://scholarsjunction.msstate.edu/td/233 This Graduate Thesis - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Automated Template A: Created by James Nail 2011 V2.01 A procedure using Rotylenchulus reniformis nucleotide sequences to quantitatively measure plant-parasitic nematode infestation levels using metagenomic DNA isolated directly from soil By Kurtis C. Showmaker A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Masters of Science in Agricultural Life Sciences in the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology Mississippi State, Mississippi December 2012 Copyright by Kurtis C. Showmaker 2012 A procedure using Rotylenchulus reniformis nucleotide sequences to quantitatively measure plant-parasitic nematode infestation levels using metagenomic DNA isolated directly from soil By Kurtis C. Showmaker Approved: _________________________________ _________________________________ Gary W. Lawrence Shien Lu Associate Professor of Biochemistry, Associate Professor of Biochemistry, Molecular Biology, Entomology and Molecular Biology, Entomology and Plant Pathology Plant Pathology (Co-Director of Thesis) (Co-Director of Thesis) _________________________________ _________________________________ Vincent Klink Clarissa J. Balbalian Assistant Professor of Biological Sciences Committee Participant of Biochemistry, (Committee member) Molecular Biology, Entomology and Plant Pathology (Committee member) _________________________________ _________________________________ Michael A. Caprio George Hopper Professor of Biochemistry, Molecular Dean of the College of Agriculture Biology, Entomology and Plant Pathology Life Sciences (Graduate Coordinator) Name: Kurtis C. Showmaker Date of Degree: December 15, 2012 Institution: Mississippi State University Major Field: Agricultural Life Sciences Major Professor: Major Professor(s) Title of Study: A procedure using Rotylenchulus reniformis nucleotide sequences to quantitatively measure plant-parasitic nematode infestation levels using metagenomic DNA isolated directly from soil Pages in Study: 99 Candidate for Degree of Masters of Science Molecular diagnostic tests have been developed and utilized to diagnose and to confirm diagnoses of many plant-parasitic nematodes. We evaluate the potential of a qPCR assay to detect and quantify Rotylenchulus reniformis in Mississippi directly from soil. A novel pipeline utilizing multiple databases containing nematode DNA and EST sequences was developed to aid in the selection of R. reniformis primers used in a PCR and qPCR assays. In vitro testing showed that the primers and probes developed from the novel pipeline for the qPCR assays could accurately detect the presence of R. reniformis. Subsequent testing resulted in a trend of increasing observed number of R. reniformis resulting in increasing estimates by qPCR DEDICATION I dedicate this research to my son Chase. ii ACKNOWLEDGEMENTS The author expresses his gratitude to his committee members; Clarissa Balbalian, Shi-en Lu, Vincent Klink, and Gary Lawrence. A special thanks is given to my major professor whom taught me the how to identify plant-parasitic nematodes. iii TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ........................................................................................................... ix CHAPTER I. DEVELOPMENT OF MOLECULAR MARKERS FOR ROTYLENCHULUS RENIFORMIS ....................................................1 INTRODUCTION .............................................................................................1 Statement of Problem ...................................................................................1 Life Stage Development of Rotylenchulus reniformis .................................2 Distribution and Host Ranges of Rotylenchulus spp....................................3 The Pathogen Rotylenchulus reniformis ......................................................7 Management Practices for Rotylenchulus reniformis ..................................7 The Development of Molecular Diagnostic Tools for Nematodes ..............8 Quantitative PCR .......................................................................................12 Nematode Genomics Usefulness to Molecular Diagnostics ......................13 Thesis Objective.........................................................................................15 MATERIALS AND METHODS .....................................................................17 Procurement of Nematodes ........................................................................17 Metagenomic Nematode DNA Isolation ...................................................19 Rotylenchulus reniformis ITS1 Primer Design ..........................................19 Rotylenchulus reniformis ITS1 Primer Pair Evaluation .............................20 Rotylenchulus reniformis qPCR Primer and Probe Design .......................21 Evaluation of potential qPCR Primer Pair Specificity ...............................23 Field Nematode Sampling and Extractions for qPCR ...............................24 Metagenomic Nematode DNA Extractions for qPCR ...............................24 PCR Assays on Soil Extracted Nematodes for select qPCR Primer Pairs................................................................................................25 PCR Assays on Metagenomic DNA Isolated Directly from Soil Using the qPCR Primer Pairs .........................................................25 Quantitative PCR Probe and Instrumentation ............................................26 iv Hit 2 alt qPCR Assay Conducted on Cultured Extracted Rotylenchulus reniformis ...............................................................27 Hit 2 alt qPCR Assay Conducted on Extracted Nematodes from Field Samples .................................................................................27 Hit 7 qPCR Assay Conducted on Extracted Cultured Rotylenchulus reniformis .......................................................................................28 Hit 7 qPCR Assay Conducted on Extracted Nematodes from Field Samples ..........................................................................................29 Hit 7 qPCR Assay on Direct Soil Metagenomic DNA ..............................29 Confirmation of PCR amplification in PCR and the qPCR reactions .........................................................................................29 RESULTS ........................................................................................................30 Procurement of Nematodes ........................................................................30 Rotylenchulus reniformis ITS1 Primer Pair Evaluation .............................31 Rotylenchulus reniformis Specific PCR Primer Pair and Probe Design ............................................................................................31 Evaluation of Potential qPCR Primer Pair Specificity ..............................32 Field Nematode Sampling and Extractions for qPCR ...............................33 PCR Assays on Soil Extracted Nematodes for select qPCR Primer Pairs................................................................................................33 PCR Assays on Metagenomic DNA Isolated Directly from Soil using the qPCR Primer Pairs..........................................................34 Hit 2 alt qPCR Assay Conducted on Cultured Extracted Rotylenchulus reniformis ...............................................................35 Hit 2 alt qPCR Assay Conducted on Extracted Nematodes from Field Samples .................................................................................35 Hit 7 qPCR Assay Conducted on Extracted Cultured Rotylenchulus reniformis .......................................................................................35 Hit 7 qPCR Assay Conducted on Extracted Nematodes from Field Samples ..........................................................................................36 Hit 7 qPCR Assay on Direct Soil Metagenomic DNA ..............................36 Confirmation of PCR Amplification in PCR and the qPCR Reactions ........................................................................................37