Thesis (doctoral)--Swedish University of Agricultural Sciences, 2003. / Appendix consists of reprints and manuscripts of five papers co-authored with others. Includes bibliographical references. Also partially available electronically via World Wide Web in PDF format; online version lacks appendix.

We constructed a cDNA library with mRNA isolated from Sf9 cells infected with Spodoptera littoralis nucleopolyhedrovirus type B (SpliNPV-B) and identified the lef-3 gene from this library. Northern blot analysis showed that SpliNPV-B lef-3 mRNA was expressed as a 1.6 Kb transcript at 5 hours post infection (p.i.), reached high levels at 24 hours p.i., and remained highly expressed at 56 hours p.i.. Transcriptional mapping showed that lef-3 transcription started from two initiation sites (the distal and the proximal transcription initiation sites) located approximately 9 nucleotides apart. The sequences that modulate lef-3 expression were investigated by transient expression assays using a reporter gene under transcriptional control of the lef-3 promoter. Deletion analysis of the 5'-flanking region demonstrated that sequences up to 584 bases 5' of the distal transcription initiation site affected the level of reporter activity, indicating that this region contains transcription regulators. A region that was sufficient to direct basal level of promoter activity, the minimal promoter, was identified. This region encompasses the two transcription initiation sites, two TATA boxes, and a GATA motif. Mutations in the GATA motif resulted in substantial decrease in the level of reporter activity, suggesting that the GATA motif is an important element in the regulation of lef-3 gene expression. The sequence of a 2.6-kb region (mu 42.8-46.8) encompassing the lef-3 gene and flanking sequences was determined. Alignment of the predicted amino acid sequence of the LEF-3 polypeptide of SpliNPV-B with the putative sequences of AcMNPV and OpMNPV LEF-3 revealed low levels of sequence conservation (26% and 21% amino acid sequence identity, respectively). This low level of sequence conservation corroborates the view that, within the genus Nucleopolyhedrovirus, SpliNPV-B is distantly related to AcMNPV and OpMNPV. / Graduate

Baculoviruses are viruses of arthropods with large rod-shaped virions that contain supercoiled double-stranded DNA genomes. These viruses have been used as gene expression vectors and insect biological control agents, and have been studied as a virus model for the investigation of molecular mechanisms, such as apoptosis, gene expression, DNA replication, and virus-host interaction. Our current knowledge about baculovirus is largely based on the studies of the Autographa californica ucleopolyhedrovirus and the closely related species. In spite of the increasing interest of recombinant baculoviruses as gene expression and delivery vectors and bioinsecticides, the mechanisms of baculovirus DNA replication and virus-host interaction are still poorly understood. To take advantage of baculovirus diversity and their specific host-ranges, I studied the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV). Previous investigations indicated that SpliNPV possesses a unique host-range and genetic organization. In this dissertation, I studied the SpliNPV infection of an orthopteran cell line derived from the grasshopper, Melanopus sanguinipes, and provided evidence of viral DNA replication and production of viable virus progeny. I next investigated SpliNPV infection in five cell lines derived from three lepidopteran families: Sf9, CLS79 and Se1 cell lines from Spodoptera (Noctuidae), Ld652Y cells from Lymantria dispar (Lymantriidae), and Md210 cells from Malacosoma disstria (Lasiocampidae), which represented permissive (Sf9, CLS79, and Se1), semipermissive (Ld652Y), and non-permissive (Md210) cell lines. SpliNPV infection in permissive cell lines resulted in viral gene expression, DNA replication, and production of viable progeny. While the semi-permissive cell line displayed reduced and delayed transcription of viral genes and supported limited viral DNA replication, the non-permissive cell line displayed dramatically reduced viral transcription and abolished viral progeny. Transient expression assays using SpliNPV early- or very late-promoter reporters suggested that non-productive infection of SpliNPV in semi- or non-permissive cell lines was a consequence of limited viral specific transcription at the early phase of viral infection. Having documented the infection events in these cell lines, I investigated the mechanism of SpliNPV DNA replication. Using transient replication assays I have identified a non-hr origin of SpliNPV DNA replication. With limited sequence similarity to other NPV non-hr origins, the putative SpliNPV origin consists of sequence motifs found in other origins of virus DNA replication, such as imperfect palindromes, direct repeats, and transcription factor binding sites. Transient expression assays indicated that the putative non-hr origin represses the SpliNPV early gene, lef-3. Gel mobility shift analyses confirmed that nuclear proteins from both infected and uninfected cells bound with specificity to the putative origin. After identification and characterization of the cis-acting factor involved in viral DNA replication, I then identified a trans-acting factor involved in viral DNA replication. I have sequenced a 6.4 kb DNA from SpliNPV genome that contains an ORF encoding a predicated polypeptide of 998 amino acid sequences. Comparative sequence analyses demonstrated that the ORF encoded a DNA polymerase (dnapol) that consists of conserved exonuclease domains and DNA polymerase motifs found in other eukaryotic DNA viruses and in cellular DNA polymerases. The transcription initiation site of the 3 kb SpliNPV dnapol transcript was mapped to an NPV early promoter element, ACGT. The transcript terminated at the polyadenylation signal AATAAA. Using E. coli and baculovirus expression systems, I over-expressed a 110 kDa full-length SpliNPV DNA polymerase (DNAPOL) and a truncated 96 kDa protein, in which the amino terminal 80 amino acids were deleted. Enzymatic analyses demonstrated that the DNA polymerase and 3'- 5’exonuclease activities are intrinsic to the SpliNPV DNAPOL. Deletion of the 80 amino acid residues at the N-terminal of the DNAPOL did not affect DNA polymerase and exonuclease activities. Replication products from single-stranded M13 DNA revealed that SpliNPV DNAPOL possesses a proccessive activity. / Graduate

Appraisals of investment in pest control are complicated by the problems of predicting events in biological systems. In this study, an attempt is made to estimate the two necessary components of pest control investment appraisal, namely: the production function (decreases in crop losses with unit increases in pest control investment), and the pest damage function (relating crop damage to changing infestation variables), for attacks of the lepidopterous larvae of Spodoptera I1ttoralis (Boisd.), on Cypriot lucerne pastures. It is suggested that at present the best technique available to farmers for controlling S. littoralis infestations is the single application of one of three insecticides of proven efficacy. Consequently, the cost of successful pest control is represented by one value for a wide range of larval densities. The pest damage function is described as a dynamic relationship between a number of changing environmental and crop variab1es and is presented in the form of a computer simulation. This incorporates some of the existing empirical data on pest consumption and pest and crop interaction as well as much of the additional data collected by the author. The damage and production functions are compared, and estimates are made of the minimum larval density at various timings in the crop growth cycle, which is sufficient to cause losses equal to the treatment costs (the economic threshold of treatment). These estimates are offered as a basis for decision making on the economic control of S. littoralis in Cypriot lucerne fields.

No description available.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Flumetsulam … is a member of the triazolopyrimidine sulfonanilide family of herbicides. It is used for pre or post-emergence broadleaf weed control in wheat, corn, barley and soybean. It has low toxicity and is readily degradable. A HPLC-UV method for the analysis of flumetsulam with the use of a surrogate and internal standard was developed and validated for soybean. The surrogate and internal standards are diclosulam and benzoic acid respectively. A LC-ESI-MS/MS confirmation method in the positive ion mode was also developed for flumetsulam. In the solvent-solvent extraction method, flumetsulam was extracted from the soybean with MeOH and the extract partitioned with a hexane:MTBE mixture (which was discarded). In the solid phase extraction and clean up method, C-18, SAX and SCX cartridges were used. Flumetsulam was extracted from the soybean as for the solvent-solvent extraction method. The solvent-solvent extraction method was used for flumetsulam determination at fortification levels of 10, 20, 50 and 100 ng -g soybean -1. The method has average absolute recoveries of 80% (range 72 to 90%). The absolute precision of recovery is about 16% and the detection limit is 4.8 ng g-1.The solid phase extraction method was used for flumetsulam determination at fortification levels of 20, 50, and 100 ng (ng soybean )-1. The precision of recovery is about 11%. The detection limit is 7.2 ng g-1. The MRL for flumetsulam recommended by the Australian Pesticides and Veterinary Medicines Authority is 50 ng g-1 for pulses. / Doctor of Philosophy (PhD)

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv. / Härtill 5 uppsatser.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-01-26T13:50:02Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CorrelacaoMetalobicaFungos.pdf: 2029628 bytes, checksum: 8feab0d533f4d04b3afb09538d403b20 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-01-27T13:25:47Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CorrelacaoMetalobicaFungos.pdf: 2029628 bytes, checksum: 8feab0d533f4d04b3afb09538d403b20 (MD5) / Made available in DSpace on 2017-01-27T13:25:47Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_CorrelacaoMetalobicaFungos.pdf: 2029628 bytes, checksum: 8feab0d533f4d04b3afb09538d403b20 (MD5) Previous issue date: 2016-06-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os fungos endofíticos são uma fonte promissora de metabólitos secundários com aplicações biotecnológicas. Esses micro-organismos habitam o interior dos tecidos vegetais sem causar nenhum dano ao hospedeiro e como fruto dessa interação podem produzir algumas das substâncias sintetizadas pelas plantas hospedeiras. Neste contexto o uso de fungos endofíticos como fonte de biomoléculas em substituição as de planta representa vantagens econômicas e ambientais. Espécies da família Amaryllidaceae produzem alcaloides e outros metabólitos com atividades biológicas. Entre estas espécies destacam-se Crinum americanum L. E Hymenocallis littoralis (Jacq.) Salisb. No entanto, não há dados sobre fungos endofíticos de espécies da família. Diante dos exposto o objetivo deste trabalho foi investigar o potencial biotecnológico de fungos endofíticos de C. americanum e H. littoralis em comparação com as plantas hospedeiras e estudar a interação entre endofíticos e a planta hospedeira. Como resultados foram isolados 94 fungos endofíticos das duas espécies de Amaryllidaceae investigadas, dos quais 49 foram identificados e pertencem aos gêneros Colettotrichum, Acremonium, Trichoderma e Fusarium. Constatou-se que do total das linhagens analisadas 56 apresentaram lipídios em seus extratos, 21 cumarinas, 29 anronas e 2 apresentaram alcaloides. Foram selecionadas 12 linhagens de fungos endofíticos que apresentaram os melhores resultados na detecção de classes de metabólitos e os extratos foram analisados por Cromatografia em Camada Delgada e por Cromatografia Gasosa acoplada a Espectrometria de Massas em comparação com os extratos metanólicos das plantas hospedeiras. Constatou-se a correlação ente as classes de metabólitos detectados nos extratos dos fungos endofíticos e das plantas hospedeiras, indicando que estes micro-organismos são capazes de produzir algumas das mesmas substâncias que as plantas hospedeiras e a avaliação da atividade antimicrobiana destacou alguns extratos com atividade contra Candida parapsilosis. O extrato metanólico das folhas de H. littoralis e do fungo endofítico MIBA 0796 apresentaram os resultados mais relevantes com percentual de inibição acima de 60% contra a levedura. Com o estudo da interação entre fungos endofíticos de C. americanum e a planta hospedeira pode-se constatar que os micro-organismos ocorrem no interior das células vegetais e em condições favoráveis se desenvolveram e ocuparam também os espaços intercelulares. Esta localização dos endofíticos nos tecidos vegetais pode facilitar a troca de material genético entre o vegetal e os micro-organismos o que explicaria a correlação metabólica constatada nesta pesquisa. / The endophytic fungi are a promising source of secondary metabolites with biotechnological applications. These microorganisms inhabit the interior of plant tissues without causing any damage to the host and as a result of this interaction can produce some of the substances synthesized by the host plant. In this context the use of endophytic fungi as a source of biomolecules in the replacement of plant represents economic and environmental advantages. Species in the Amaryllidaceae family produce alkaloids and other metabolites with biological activities. Among these species include Crinum americanum L. and Hymenocallis littoralis (Jacq.) Salisb. However, no data on endophytic fungi from species of the family were found. In the view of the above stated the aim of this work was to scrutinize the biotechnological potential of endophytes of C. americanum and H. littoralis as compared to host plants and to study the interactions between endophytes and host plant. As results 94 endophytic fungi were isolated from two species of the investigated Amaryllidaceae, of which 49 were identified and belonging to the Colettotrichum class, Acremonium, Trichoderma and Fusarium. It was noted that of the total strains tested 56 showed lipids in its extracts, 21coumarins, 29 anthrones and 2 showed alkaloids. 12 strains of endophytic fungi were selected which presented the best results in the detection of classes of metabolites and the extracts were analyzed by Thin-layer chromatography and by Gas chromatography-mass spectrometry in comparison with the methanolic extracts of host plants. It was noted the correlation between the classes of metabolites detected in extracts of the endophytic fungi and host plants, indicating that these micro-organisms are able to produce some of the same substances that the host plants and the assessment of antimicrobial activity highlighted some extracts with activity against Candida parapsilosis. The methanolic extract of leaves of H. littoralis and endophytic fungus MIBA 0796 presented the most relevant outcomes with percentage of inhibition above 60% against yeast. With the study of the interaction between C. americanum endophytes and the host plant it can observe that microorganisms act within the plant cells and in favorable conditions develop and occupy intercellular spaces as well. This location of the Endophytes in plant tissues may facilitate the exchange of genetic material between plants and microorganisms explaining how metabolic correlation found in this research.

No description available.