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Moleculartoolsto Identifyand Study Invasivepests in Brazil

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Moleculartoolsto Identifyand Study Invasivepests in Brazil Molecular tools to identify and study invasive pests in Brazil Jonas André Arnemann Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences at the faculty of Bioscience Engineering of Ghent University, with co-tutelage with the Post-Graduate Program of the Federal University of Santa Maria I dedicate this thesis to my parents Nelson and Helena, and to my brother Jardel. “Be kind; everyone you meet is fighting a hard battle.” John Watson “Talvez não tenha conseguido fazer o melhor, mas lutei para que o melhor fosse feito. Não sou o que deveria ser, mas Graças a Deus, não sou o que era antes.” Marthin Luther King Promotors: Prof. dr. ir. Guy Smagghe Department of Crop Protection Faculty of Bioscience Engineering Ghent University, Ghent, Belgium Prof. Dr Jerson V. Carús Guedes Department of Entomology Federal University of Santa Maria Santa Maria, Rio Grande do Sul, Brazil Chair of the examination committee: Prof. Dr. Élgion Lúcio da Silva Loreto Department of Biology Federal University of Santa Maria Santa Maria, Rio Grande do Sul, Brazil Members of the examination committee: Prof. Dr. Rodrigo J. Seminoti Jacques Department of Soil Science Federal University of Santa Maria Santa Maria, Rio Grande do Sul, Brazil Dr. Wee Tek Tay, PhD Senior Research Scientist CSIRO Health and Biosecurity Flagship Canberra, ACT, Australia Prof. Dr. Juliano Tomazzoni Boldo Department of Biology Federal University of Pampa-Unipampa São Gabriel, Rio Grande do Sul, Brazil Prof. Dr. ir. Peter Bossier Department of Animal Production Faculty of Bioscience Engineering Ghent University, Ghent, Belgium Dean: Prof. dr. ir. Guido van Huylenbroeck Rector: Prof. dr. Anne De Paepe Dutch translation of the title: Het gebruik van moleculaire tools voor de identificatie van nieuwe invasieve plaagorganismen in Brazilië. Portuguese translation of the title: Uso de ferrmentas moleculares para identificar e estudar pragas invasivas no Brasil. Front cover: Top: xxx Please refer to this work as follows: Arnemann, JA. (2015). Molecular tools to identify and study invasive pests in Brazil. PhD Thesis. Ghent University, Ghent, Belgium. ISBN: 978-90-5989-841-7 This study was financially supported with grants from the CAPES, UFSM and CSIRO. The author and the promoters give the permission to use this thesis for consultation and to copy parts of if for personal use. Every other use is subject to the copyright laws; more specifically the source must be extensively specified when using results from this thesis. Table of Contents CHAPTER I MOLECULAR TOOLS TO IDENTIFY AND STUDY INVASIVE PESTS IN CONSE SUL OF AMERICA ... 1 1.1 Biosecurity, food security and agriculture .............................................................. 2 1.2 Invasive pests in South America and Brazil ............................................................ 4 1.3 The Soybean Stem Fly (SSF), Melanagromyza sojae (Zehntner) ............................ 7 1.4 Helicoverpa armigera occurrence in Brazil ............................................................. 9 1.5 Mitochondrial DNA markers for pest studies ....................................................... 11 1.6 Using molecular tools to help confirm invasive pests and study population dynamics ..................................................................................................................... 13 1.7 References ............................................................................................................ 15 CHAPTER II COMPLETE MITOCHONDRIAL GENOMES OF THE SOYBEAN STEM FLY (SSF) MELANAGROMYZA SOJAE (D IPTERA : AGROMYZIDAE ) ........................................................... 26 2.1 Introduction .......................................................................................................... 27 2.2 Material and Methods .......................................................................................... 30 2.2.1 Morphological identification .......................................................................... 31 2.2.2 Molecular characterization of the complete mitochondrial DNA genome ... 31 2.2.3 Intra-species nucleotide diversity in M. sojae ............................................... 32 2.2.4 SSF mtDNA PCR markers development and PCR optimisation ...................... 32 2.2.5 Inter-species nucleotide diversity in Agromyzidae ........................................ 34 2.2.6 Phylogeny of Agromyzidae based on partial mtCOI gene.............................. 34 2.3 Results ................................................................................................................... 34 2.3.1 Species confirmation by morphology ............................................................ 34 2.3.2 Molecular characterization of complete mitochondrial DNA genome.......... 35 2.3.3 Intraspecies mitogenome PCG SNP patterns and nucleotide diversity ......... 40 2.3.4 PCR primers design......................................................................................... 42 I 2.3.4 Inter-species diversity and maximum Likelihood phylogeny analysis of M. sojae and related Agromyzidae flies ....................................................................... 43 2.4 Discussion.............................................................................................................. 46 2.5 References ............................................................................................................ 49 CHAPTER III GENETIC DIVERSITY OF MELANAGROMYZA SOJAE (D IPTERA : AGROMYZIDAE ) IN SOYBEAN (GLYCINE MAX ) IN BRAZIL ................................................................................................ 0 3.1 Introduction .......................................................................................................... 57 3.2 Material and methods .......................................................................................... 61 3.2.1 Samples .......................................................................................................... 61 3.2.2 Total genomic DNA (gDNA) extraction ........................................................... 64 3.2.3 Primers, PCR amplification and sequencing of partial mitochondrial DNA COI gene ......................................................................................................................... 65 3.2.4 Sequence analysis and molecular characterization of mtDNA COI gene ...... 66 3.2.5 MtDNA haplotypes network and COI phylogenetic analysis ......................... 67 3.3 Results ................................................................................................................... 67 3.3.1 Species confirmation ...................................................................................... 67 3.3.2 PCR amplification and sequence analysis ...................................................... 68 3.3.3 Haplotypes patterns ....................................................................................... 68 3.4 Discussion.............................................................................................................. 74 3.5 References ............................................................................................................ 80 CHAPTER IV MOLECULAR DETECTION AND GENETIC DIVERSITY OF THE OLD WORLD COTTON BOLLWORM HELICOVERPA ARMIGERA (L EPIDOPTERA : NOCTUIDAE ) IN CONE SUL OF AMERICA ........ 92 4.1 Introduction .......................................................................................................... 93 4.2 Materials and methods ......................................................................................... 98 4.2.1 Samples .......................................................................................................... 98 4.2.2 Total genomic DNA (gDNA) extraction ........................................................... 99 II 4.2.3 Primers, PCR amplification and sequencing of partial mitochondrial DNA COI gene ......................................................................................................................... 99 4.2.4 Sequence analysis of partial mtDNA COI gene ............................................ 100 4.2.5 MtDNA COI haplotype network and evolutionary divergence .................... 101 4.2.6 Analysis of Helicoverpa armigera haplotypes distributions ........................ 103 4.2.7 Null and alternative hypotheses .................................................................. 104 4.2.8 Test statistics used ....................................................................................... 104 4.2.9 Null model .................................................................................................... 105 4.2.10 Calculating P-values ................................................................................... 106 4.2.11 Validating the null model ........................................................................... 106 4.2.12 Sub-table analysis ....................................................................................... 107 4.2.13 Brazil vs. Non-Brazil .................................................................................... 107 4.3 Results ................................................................................................................. 108 4.3.1 PCR amplification and sequence analysis .................................................... 108 4.3.2 Haplotypes ..................................................................................................
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