A thesis submitted to the Faculty of Science under the school of Molecular and Cell Biology in fulfilment for requirements for Doctor of Philosophy Degree. February 2016 / The use of synthetic chemical pesticides has several negative implications for the Agricultural industry, which include the development of resistance to the insecticides, crop contamination and the killing of non-target insects. This has brought many scientists in the field of nematology and entomology to investigate biological control agents which can help solve identified challenges and these biocontrol agents have also included entomopathogenic nematodes. The majority of entomopathogenic nematodes species that have been isolated belong to Heterorhabditids and Steinernematids which act as vectors for insect pathogenic bacteria species belonging to the genera, Photorhabdus and Xenorhabdus, respectively. However, other species of nematodes, one of which includes a strain of Caenorhabditis briggsae, have also been shown to act as a vector for an insect pathogenic strain of Serratia marcescens. Oscheius sp. TEL-2014 EPNs have been observed to act as vectors for insect pathogenic bacteria belonging to the genus Serratia. In this study a novel insect pathogenic Serratia sp. strain TEL was isolated from the gut of infective juveniles belonging to a species of Oscheius sp. TEL-2014. Next generation sequencing of the bacteria was conducted by generating genomic DNA paired-end libraries with the Nextera DNA sample preparation kit (Illumina) and indexed using the Nextera index kit (Illumina). Paired-end (2 × 300 bp) sequencing was performed on a MiSeq Illumina using the MiSeq reagent kit v3 at the Agricultural Research Council Biotechnology Platform. Quality control and adapter trimming was performed and the genome was assembled using SPADES. 19 contigs were generated with an average length of 301767 bp and N50 of 200,110 bp. The genome of the Serratia sp. TEL was found to be 5,000,541 bp in size, with a G+C content of 59.1%, which was similar to that of other Serratia species previously identified. Furthermore, the contigs were annotated using NCBI Prokaryotic Genome Automatic Annotation Pipeline. Features of the annotated genome included protein encoding sequence or genes, rRNA encoding genes, tRNA encoding genes, ncRNA sequences and repeat regions. 4,647 genes were found and 4,495 were protein-coding sequences (CDS). The genome contains 36 pseudo genes, 2 CRISPR arrays, 13 rRNA genes with five operons (5S, 16S, 23S), 88 tRNAs genes, 15 ncRNA sequences and 9 frameshifted genes. Several genes involved in virulence, disease, defense, stress response, cell division, motility and chemotaxis were identified. This genome sequence will allow for the investigation of identified genes and that will be critical in furthering the understanding of the insect pathogenicity of Serratia sp. strain TEL. Furthermore, it will provide additional genomic insights about the insect-nematode interactions and thus help us improve their ability to be used as biological control agents in agricultural industries. Oscheius sp. TEL-2014 was tested for its entomopathogenicity and it was found that this species was able to infect and kill two model insects Galleria mellonella and Tenebrio molitor. This new nematode species brought 100% mortality within 72 h post-exposure in G. mellonella and whereas, within 96 hours in T. molitor. Following morphometrics analysis of Oscheius sp. TEL-2014 it was concluded that this nematode is described as a novel entomopathogenic nematode species based on its morphometrics and 18S rRNA gene sequence originality. Whole genome sequencing of Oscheius sp. TEL-2014 inbred lines (7 and 13) was performed using Illumina Hiseq sequencing system and paired ends library preparation protocol. Sequencing reads assembled on Velvet resulted in generation of 75965 contigs (line 7) and 53190 contigs (line 13). Gene prediction tools showed that proteins involved in gene expression and DNA replication are present in Oscheius sp. TEL-2014. The draft genome of Oscheius nematodes will support the improvement and initiation of further studies intended to help us understand the molecular and metabolic processes in this genus.

Genetically modified organisms are entering the human diet in all over the world. In order to have transparency in the foods that are being consumed, there is a need to trace the genetically modified organisms (GMOs) in the market and consequently this need brings the necessity of analytical methods that are capable of detecting, identifying and quantifying the transgenic events. These analytical methods also form the basis of the labeling regulations that are tried to be formed regarding GMOs. The main aim of this study is to develop and apply the detection methods for the two of the tomato events, delayed ripening and insect resistant. Currently the only validated detection methods are mainly for the corn, soybean, and cotton. There is no validated detection method for tomato. Tomato is one of the most consumed food products in Turkey and it is also among the controversial organisms in terms of genetic modifications and labeling, therefore the analysis of the genetic modifications in tomato is crucial. In this study, DNA-based detection is performed, with PCR being the chosen method of study. In order to detect the GMO-derived DNA, the method of analysis includes the following studies: species-specific, screening, gene-specific, construct-specific and inverse PCR. In addition, the quantification method is developed using the real-time PCR. In order to develop the procedure of identification method, the reference samples are used and the unknown varieties that are to be analyzed using this method are expected to have similarities with the authorized transgenic events.

Tomato, which is one of the most important component of human diet, has been genetically modified to develop some properties like delayed ripening and insect resistance. In order to give a choice to the consumer, it is necessary to detect and label GM foods. This study was carried out to detect genetically modified tomato samples purchased from different food markets of Turkey. PCR method was used to detect genetically modified insect resistant tomatoes. The DNAs of collected samples were isolated according to CTAB DNA extraction protocol and the amplification capacity of isolated samples were checked with patatin specific control PCR. Screening tests of tomatoes were done by targeting 35S promoter, Nos terminator and NptII kanamycin resistance gene with four primer sets. It was aimed to detect Cry1A and Cry1Ac genes with three PCR systems, in order to identify insect resistant samples. From twenty-eight samples, twenty-two gave positive amplification signal in NptII specific PCR system and results were confirmed with sequence analysis. Additionally, we observed seventeen and ten DNA fragments with Cry1A-F/Cry1A-R and Cry1Ac-F/Cry1Ac-R primer sets respectively, it is necessary to confirm these results with DNA sequencing.

This study was carried out to analyze tomato samples and tomato seeds, purchased from different food markets of Turkey randomly, for the presence of genetic modification by using PCR method as it allows more specific detection. The DNAs of collected samples were isolated according to CTAB DNA extraction protocol and also with extraction kits. Screening tests of tomatoes were done by targeting 35S promoter, NOS terminator and NptII kanamycin resistance gene with eight different primer sets. Real time PCR is used to confirm 35S and NOS positives results obtained from conventional PCR. In this study, it was observed that 14 out of 35 seed samples, and 14 out of 40 fresh tomato samples which were screened had at least one transgenic element of 35S promoter, NOS terminator and NPTII kanamycin resistance gene indicating the possible presence of genetic modifications. After screening, gene specific studies were carried out for PG, sam-k indicating F type ripening delayed tomato and the 35 1 N lines respectively and cry1Ac genes inserted in 5345-1 insect resistant tomato line. PG and sam-k specific primers were not amplified in any of the samples investigated whereas 18 out of 75 samples were cry1Ac positive and 1 out of 75 samples was sam-k positive. Positives were confirmed by sequence analysis. Additionally, construct specific primers specific to 5345-1 and 35 1 N lines were designed. PCR amplicons indicate the existence of the construct sequence. In order to verify the results, PCR products were sent to sequence analysis

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-08-26T14:45:40Z No. of bitstreams: 1 Wellington Marques da Silva.pdf: 838955 bytes, checksum: 5c601201dc688914f0da042a97074570 (MD5) / Made available in DSpace on 2016-08-26T14:45:41Z (GMT). No. of bitstreams: 1 Wellington Marques da Silva.pdf: 838955 bytes, checksum: 5c601201dc688914f0da042a97074570 (MD5) Previous issue date: 2015-02-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Tuta absoluta is a key pest of tomato crops and the use of insecticides is still the main method of control. However, overuse of them has contributed to the development of resistant populations. Although the pyrethroid resistance mechanism in this species has been described, there is no information on their status in Brazilian populations. Regarding the spinosyns, despite the reports of resistance to these molecules, the underlying mechanisms are unknown. The objective of this research was to characterize the resistance to pyrethroids and spinosad in Brazilian populations of T. absoluta by conventional methods (bioassays), enzymatic and molecular assays. All populations tested were resistant to pyrethroids. The GSTs activity and cytochrome P450-mediated N-demethylation was significantly correlated with the level of resistance to deltamethrin and permethrin suggesting that these enzymes can play a role in resistance. TaqMan assays demonstrated that the mutation in L1014F sodium channel is fixed in all populations and is associated with other mutations T929I and M918T. Spinosad synergism (synergistic with PBO, DEF, and DEM) indicated that the metabolic enzymes are not involved in resistance. Comparison of the nucleotide sequence of the subunit of the nicotinic receptor α6 T. absoluta susceptible and resistant to the point spinosad allowed identification nucleotide change resulting in the substitution of a glycine (G) in the susceptible insects by glutamic acid (E) resistant insects (G275E). The TaqMan assays revealed that the frequency of the resistant allele in the population is low. The diagnostic dose bioassays correlated with the resistant allele frequency. The T. absoluta Brazilian populations are resistant to pyrethroid insecticides and the main mechanism of resistance is the L1014F mutation, while for spinosad insecticide, most people are sensitive and the G275E mutation can lead to the loss of efficiency of this product for control of this plague. / A Tuta absoluta é uma praga importante para a cultura do tomateiro e o uso de inseticidas ainda é principal método de controle. No entanto, a utilização indiscriminada destes tem contribuído para seleção de populações resistentes. Embora o mecanismo de resistência à piretroides nessa espécie tenha sido descrito, não há informações sobre seu status em populações brasileiras. Com relação a espinosinas, apesar dos relatos de resistência a estas moléculas, os mecanismos envolvidos são desconhecidos. O objetivo desta pesquisa foi caracterizar a resistência a piretroides e espinosade em populações brasileiras de T. absoluta por métodos convencionais (bioensaios), ensaios enzimáticos e moleculares. Todas as populações testadas foram resistentes a piretroides. A Atividade de GSTs e citocromo P450 mediado por N-desmetilação correlacionou-se significativamente com o nível de resistência a deltametrina e permetrina sugerindo que estas enzimas podem desempenhar um papel na resistência. Ensaios TaqMan demonstraram que a mutação L1014F no canal de sódio está fixa em todas as populações e foi associada com outras mutações M918T e T929I. Sinergismo de espinosade com PBO, DEF, DEM indicou que as enzimas metabólicas não estão envolvidas na resistência. A comparação da sequência de nucleotídeos da subunidade α6 do receptor nicotínico de T. absoluta suscetíveis e resistentes a espinosade possibilitou a identificação de alteração pontual de nucleotídeo, resultando na substituição de uma glicina (G) nos insetos suscetíveis por um ácido glutâmico (E) em insetos resistentes (G275E). Os ensaios de TaqMan revelaram que a frequência do alelo resistente está baixa nas populações. Os bioensaios de dose diagnóstica correlacionaram com frequência do alelo resistente. As populações brasileiras de T. absoluta são resistentes aos inseticidas piretroides e o principal mecanismo de resistência é a mutação L1014F, enquanto para o inseticida espinosade, a maioria das populações são sensíveis e a mutação G275E pode acarretar a perda da eficiência deste produto.