Orientador: Marcelo Agenor Pavan / Coorientador: Renate Krause Sakate / Banca: Marcio Martinello Sanches / Banca: Valdir Atsushi Yuki / Banca: Julio Massaharu Marubayashi / Banca: Antonio Carlos Maringoni / Resumo: Espécies de vírus dos gêneros Potyvirus, Carlavirus e Allexivirus são comumente encontradas na cultura do alho (Allium sativum L.) e em decorrência da sua propagação vegetativa, há o acúmulo de vírus de um ciclo para outro, formando um complexo entre espécies dos gêneros citados. Uma das principais causas da redução da produtividade ocorre devido à infecção por vírus, e a termoterapia associada à cultura de tecido pode ser um método eficiente para limpeza clonal e obtenção de alho semente livre de vírus. O estudo da degenerescência do alho livre de vírus é, portanto, importante para se conhecer o número de ciclos em que o alho semente, inicialmente sadio, pode ser multiplicado no campo, sem que ocorra a redução da produtividade. Neste estudo foi verificado que após três anos a produtividade foi 13,75% superior ao alho 100% infectado por vírus. Os bulbilhos aéreos também podem ser utilizados para a multiplicação de sementes e tem como vantagem o baixo custo quando comparado à utilização de bulbos provenientes da cultura de tecido. Entretanto, essa técnica só se torna viável com a utilização de matrizes isentas de vírus, pois a transmissão de vírus para os bulbilhos aéreos, provenientes de matrizes infectadas, podem chegar a 83,33% no caso dos potyvirus, 20% para carlavirus e 70% para allexivirus, segundo dados obtidos neste trabalho. Realizar uma diagnose precisa dos vírus que infectam o alho torna-se essencial para que não ocorram falhas durante a indexação. Pôde-se observar que para a detecção dos potyvirus, utilizando o teste de ELISA indireto, deve-se utilizar folhas novas, entre 21 e 63 dias após o plantio, pois a concentração viral nesse período é a mais elevada. Diferentes técnicas como o DIBA colorimétrico, DIBA quimioluminescente, ELISA e PCR em tempo real foram avaliadas para detecção do LYSV, aos 21 dias após a inoculação ... / Abstract: Virus species of genera Potyvirus, Carlavirus and Allexivirus are commonly infecting garlic plants (Allium sativum L.). Due to the vegetative propagation, the accumulation of viruses might occur from one cycle to another. The infection of viruses might induce yield losses, and the technique of thermotherapy associated with meristem tip culture is an efficient method to obtain virus-free seeds. The study of degeneration of seeds free of virus is important to know the number of cycles in which garlic may be multiplied in the field without reduction of productivity. In this study it was observed that after three years, the productivity increased 13,75% compared to 100% infected seeds. Currently, aerial bulbils may be used for multiplication of seeds with low cost, compared to meristem tip culture. However, this is a viable technique only if the matrix is free of viruses, because the transmission to aerial bulbils, from infected plants can reach 83.33 % for potyviruses, 20% for carlavirus and 65% for allexivirus, data obtained in this work. An accurate diagnosis in viruses that infect garlic is important to avoid mistakes during indexing material. It was 4 observed that the best time to detect potyvirus, through ELISA test is between 21 and 63 days after planting on young leaves, as the virus concentration is higher in this period. Whem using different techniques, such as the colorimetric DIBA, chemiluminescent DIBA, ELISA, and real-time PCR, it was possible to perform the detection of LYSV 21 days after inoculation. ELISA test detected LYSV only at 21 days after inoculation, whereas using real time PCR, in 5 days after inoculation it was possible to detect the virus due to its high sensitivity. The other tests also detected the virus 21 days after infection / Doutor

Orientador: Marcelo Agenor Pavan / Coorientador: Renate Krause Sakate / Banca: Julio Massaharu Marubayashi / Banca: Rumy Goto / Resumo: A mandioquinha-salsa (Arracacia xanthorrhiza Bancroft) é uma hortaliça originária da região andina, Venezuela, Colômbia, Equador, Peru e Bolívia. A família das apiáceas compreende também outras hortaliças importantes, como cenoura, salsa, coentro, aipo, dentre outras. A mandioquinha-salsa é propagada de forma vegetativa através dos propágulos, e isso pode favorecer a ocorrência de um número considerável de patógenos que causam degenerescência, principalmente os vírus. Está comprovado que alguns vírus são fator limitante em culturas de propagação vegetativa. Portanto, o presente trabalho teve como objetivos avaliar por meio de métodos sorológico e molecular 241 amostras oriundas de diferentes localidades, a fim de fazer um estudo de ocorrência e predominância das espécies virais e verificar a variabilidade biológica dos isolados encontrados por meio de transmissão por extratos vegetais. A detecção foi realizada através de Enzyme linked immunossorbent assay (ELISA) utilizando antissoro comercial anti-poty para o gênero Potyvirus. Para a análise da variabilidade, foram desenhados 2 oligonucleotídeos para amplificar a região codificadora da proteína capsidial para o gênero Cheravirus, e para a detecção de potyvirus e nepovirus, oligonucleotídeos foram obtidos da literatura. No estudo foi possível detectar a espécie Arracacha motlle virus do gênero Potyvirus, coletadas nos estados de Goiás, Minas Gerais, Brasília e Paraná. Os demais vírus testados não foram identificados em nenhuma das amostras analisadas. Além desses testes de identificação, ainda foi realizada microscopia eletrônica de transmissão em seis amostras, das quais duas foi possível a identificação de potyvirus. O teste de gama de hospedeiras foi realizado e mostrou que o AMoV tem gama de hospedeiras bastante restrita, sendo possível infectar somente três de nove espécies testadas ... / Abstract: Arracacha (Arracacia xanthorrhiza Bancroft) is a vegetable crop of the Andean region, formed by Venezuela, Colombia, Ecuador, Peru and Bolivia. The Apiacea family also have other important vegetable crops such as carrots, parsley, cilantro, celery, among others. Arracacha is propagated vegetatively and this can favor the occurrence of a considerable number of pathogens that cause degeneration, especially viruses. Some viruses are a limiting factor in vegetatively propagated crops. Therefore, the present study aimed to evaluate by serological and molecular methods 241 samples from different localities in order to verify the occurrence and predominance of the viruses species and verify the biological variability by sap inoculation. The detection was performed using enzyme-linked immunossorbent assay (ELISA) technique, using commercial antiserum against-potyvirus. For variability analysis, primers were designed to amplify the coding region of the coat protein for the 4 genus Cheravirus, and oligonucleotides were obtained from literature for the detection of the genus potyvirus and nepovirus, . It was possible to detect Arracacha mottle virus, from genus Potyvirus, in the states of Goiás, Minas Gerais, Brasilia and Paraná. The others viruses tested were not identified in the collected samples. Six samples were analysed by electron microscopy, and the potyvirus were identified only in two samples. The host range was restricted, infecting only three species of nine tested. The nucleotide identity ranged from 96% to 99%, between collected samples and sequence from GenBank (acess DQ925486), showing low genetic variability among them / Mestre

Made available in DSpace on 2016-04-28T14:57:34Z (GMT). No. of bitstreams: 1 2008 - Everaldo Hans Studt Klein.pdf: 5165097 bytes, checksum: ccfbfbc6baec84bc829dad886555adf9 (MD5) Previous issue date: 2008-07-15 / It was conducted a survey of the major diseases that occur in orchids in the State of Rio de Janeiro, through collection of plants in various localities in the period April 2006 to June 2008. Fifty-three plants had their diseases and pathogens identified, of which 35.9% were infected by fungi, being 17% by Fusarium oxysporum, 13.2% by Colletotrichum gloeosporioides, 1,9% by Botrytis sp., 1,9% by Puccinia sp. and 1.9% by Phyllosticta capitalensis. 51% of the plants were infected by virus with simple and double infections of Cymbidium mosaic virus - CymMV and Odontoglossum ringspot virus - ORSV. 1.9% were infected by the bacterium Pectobacterium carotovorum subsp. carotovorum and 3.8% by nematodes of the genus Aphelenchoides. The nematodes Aphelenchoides fragariae and A. ritzemabosi were identified infecting hybrid orchids, which is the first report of parasitism of these nematodes in orchid and of A. ritzemabosi parasiting Asplenium nidus L. in Brazil. It also made the first record of the occurrence of Cymbidium mosaic virus - CymMV infecting Bamboo orchid Arundina bambusifolia Lindl. in the State of Rio de Janeiro. Based on the identification of pathogens of that survey, it was developed a diagnostic kit for the main diseases of orchids. Aiming to test antimycotic properties of three substances in culture medium for cultivation of orchids, using as test- plants Sophronitis cernua Lindl. and Epidendrum ellipticum Grah. Of the substances tested, only powdered cinnamon (Cinnamomum zeylanicum J. Presl), at the dose of 6 grams / liter positively influenced the germination of seeds of Epidendrum ellipticum Graham, and the addition of clove (Syzygium aromaticum) at doses of 3, 5 and 6 grams / liter caused the inhibition of in vitro germination of seeds of Epidendrum ellipticum Graham. and Sophronitis cernua (Lindl.) Hook. The addition of commercial garlic (Allium sativum L.) powder in doses of 6 and 10 grams / litre, also inhibited in vitro germination of seeds of Epidendrum ellipticum Graham. / Foi realizado levantamento das principais doen?as que ocorrem em orqu?deas no Estado do Rio de Janeiro, atrav?s de coleta de plantas em v?rias Localidades, no per?odo de abril de 2006 a junho de 2008. Cinq?enta e tr?s plantas tiveram suas doen?as e pat?genos identificados, dos quais 35,9% das plantas estavam infectadas por fungos, sendo 17% por Fusarium oxysporum, 13,2% por Colletotrichum gloeosporioides, 1,9 % por Botrytis sp., 1,9% por Puccinia sp. e 1,9% por Phyllosticta capitalensis. 51% das plantas estavam infectadas por v?rus com infec??es simples e duplas de Cymbidium mosaic virus - CymMV e Odontoglossum ringspot virus - ORSV. 1,9 % estavam infectadas pela bact?ria Pectobacterium carotovorum subsp. carotovorum e 3,8 % por nemat?ides do g?nero Aphelenchoides. Foram identificados os nemat?ides Aphelenchoides fragariae e A. ritzemabosi infectando orqu?deas h?bridas, sendo esse o primeiro relato de parasitismo desses nemat?ides em orqu?dea e de A. ritzemabosi em Asplenium nidus L., no Brasil. Foi feito tamb?m o primeiro registro da ocorr?ncia Cymbidium mosaic virus CymMV infectando plantas da esp?cie Arundina bambusifolia Lindl. no Estado do Rio de Janeiro. Com base na identifica??o dos pat?genos desse levantamento, desenvolveu-se um kit diagn?stico para os principais fitopat?genos de orqu?dea. Objetivou-se testar tr?s subst?ncias de propriedades fungist?ticas em meio de cultivo de propaga??o de orqu?deas, utilizando-se como plantas-teste Sophronitis cernua Lindl. e Epidendrum ellipticum Grah.. Das subst?ncias testadas, apenas canela em p? (Cinnamomum zeylanicum J.Presl), na dosagem de 6 gramas / litro influenciou positivamente a germina??o de sementes de Epidendrum ellipticum Graham, sendo que o acr?scimo de cravo-da-?ndia (Syzygium aromaticum) comercial picado nas doses de 3, 5 e 6 g/l causou a inibi??o da germina??o in vitro das sementes de Epidendrum ellipticum Graham. e Sophronitis cernua (Lindl.) Hook. O acr?scimo de alho (Allium sativum L.) em p? comercial nas doses de 6 e 10 g/l, tamb?m inibiu a germina??o in vitro das sementes de Epidendrum ellipticum Graham.

Xue, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 95-102). / Abstract also in Chinese. / Thesis committe --- p.2 / Statement --- p.3 / Abstract --- p.4 / Acknowledgement --- p.8 / General abbreviations --- p.10 / Abbreviations of chemicals --- p.13 / List of figures --- p.15 / List of tables --- p.16 / Table of contents --- p.17 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Impact of bacterial blight on rice production --- p.25 / Chapter 1.2 --- The plant immune system --- p.25 / Chapter 1.2.1 --- Preformed resistance --- p.25 / Chapter 1.2.2 --- PAMP triggered immunity (PTI) --- p.26 / Chapter 1.2.3 --- Effecter triggered immunity (ETI) --- p.27 / Chapter 1.2.3.1 --- R genes --- p.27 / Chapter 1.2.3.2 --- Hypersensitive responses (HR) --- p.27 / Chapter 1.2.3.3 --- Systemic acquired resistance (SAR) --- p.28 / Chapter 1.2.3.3.1 --- Salicylic acid is required for SAR establishment --- p.28 / Chapter 1.2.3.3.2 --- Involvement of lipid-based molecules in SAR signaling --- p.28 / Chapter 1.2.3.3.3 --- NPR1: the master regulator of SAR --- p.29 / Chapter 1.2.3.3.4 --- Expression of pathogenesis related (PR) genes --- p.29 / Chapter 1.2.4 --- Interaction between SA and JA --- p.29 / Chapter 1.2.5 --- Other important signaling components in plant defense responses --- p.30 / Chapter 1.2.5.1 --- G proteins --- p.30 / Chapter 1.2.5.2 --- G proteins in defense responses --- p.30 / Chapter 1.3 --- OsGAPl is a C2 (protein kinase C conserved region 2) domain harboring GTPase activating protein --- p.32 / Chapter 1.4 --- OsYchFl is a GTPase and an interacting partner of OsGAPl --- p.32 / Chapter 1.5 --- Hypothesis and objectives of this research --- p.33 / Chapter Chapter 2 --- materials and methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Chemicals and reagents --- p.39 / Chapter 2.1.2 --- Commercial kits --- p.40 / Chapter 2.1.3 --- Primers used --- p.41 / Chapter 2.1.4 --- Equipment and facilities used: --- p.47 / Chapter 2.1.5 --- "Buffer, solution, gel and medium:" --- p.47 / Chapter 2.2 --- Methods: --- p.51 / Chapter 2.2.1 --- Culture of bacterial strains --- p.51 / Chapter 2.2.2 --- Composition of medium used in this work for cultivating bacterial strains: --- p.51 / Chapter 2.2.3 --- Plant growth and treatment --- p.52 / Chapter 2.2.3.1 --- Surface sterilization of Arabidopsis thaliana seeds --- p.52 / Chapter 2.2.3.2 --- Seed germination and Arabidopsis plant growth --- p.52 / Chapter 2.2.4 --- Generation of transgenic Arabidopsis --- p.53 / Chapter 2.2.4.1 --- Agrobacterium-mediated Arabidopsis transformation --- p.53 / Chapter 2.2.5 --- Pathogen inoculation test --- p.54 / Chapter 2.2.6 --- Molecular cloning --- p.54 / Chapter 2.2.6.1 --- DNA sequencing: --- p.55 / Chapter 2.2.6.2 --- Transformation of E. coli strains: --- p.55 / Chapter 2.2.6.3 --- Transformation of Agrobacteria by electroporation --- p.55 / Chapter 2.2.7 --- DNA and RNA extraction --- p.56 / Chapter 2.2.7.1 --- Plasmid DNA extraction from bacterial cells --- p.56 / Chapter 2.2.7.2 --- Genomic DNA extraction from plant tissues --- p.56 / Chapter 2.2.7.3 --- RNA extraction from plant tissues --- p.56 / Chapter 2.2.8 --- Northern blot --- p.57 / Chapter 2.2.9 --- Subcellular localization studies --- p.58 / Chapter 2.2.9.1 --- Transformation of tobacco BY-2 cells --- p.58 / Chapter 2.2.9.2 --- Maintenance of transgenic tobacco BY-2 cells --- p.59 / Chapter 2.2.9.3 --- Confocal microscopy --- p.59 / Chapter 2.2.9.4 --- Electron microscopy --- p.59 / Chapter 2.2.10 --- Bimolecular fluorescence complementation studies (BiFC) --- p.60 / Chapter 2.2.10.1 --- Construct making --- p.61 / Chapter 2.2.10.2 --- Preparation of rice protoplasts --- p.61 / Chapter 2.2.10.3 --- PEG-mediated transfection --- p.62 / Chapter 2.2.10.4 --- Detection of protein-protein interaction --- p.62 / Chapter Chapter 3 --- Results / Chapter 3.1 --- OsGAPl interacts with OsYchFl in vivo --- p.63 / Chapter 3.1.1 --- Construction of vectors for BiFC transient assay in rice protoplasts --- p.64 / Chapter 3.1.2 --- BiFC assay in rice protoplasts revealed in vivo interaction between the OsGAPl and the OsYchFl proteins --- p.66 / Chapter 3.2.1 --- Subcellular localization of OsGAPl --- p.68 / Chapter 3.2.2 --- Localization of OsGAPl and OsYchFl in rice leaves revealed by electron microscopy --- p.70 / Chapter 3.3 --- Functional characterization of OsYchFl / Chapter 3.3.1 --- Characterization of Arabidopsis YchF1 knockdown mutant --- p.75 / Chapter 3.3.2 --- Complementation of AtYchF1 knockdown Arabidopsis --- p.77 / Chapter 3.3.3.1 --- Pathogen inoculation test --- p.80 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Significance of the project --- p.85 / Chapter 4.2 --- In vivo interaction between OsGAPl and OsYchFl --- p.86 / Chapter 4.3 --- OsGAPl is located either inside the cytosol or on the plasma membrane in transgenic tobacco BY-2 cells --- p.87 / Chapter 4.4 --- Study of wounding effect on the subcellular localization of OsGAPl and OsYchFl at whole plant level by EM --- p.88 / Chapter 4.5 --- OsYchFl functions as a negative regulator of defense responses in A.thaliana --- p.90 / Chapter 4.6 --- Conclusion --- p.92 / References --- p.95 / Appendix --- p.103

The anthracnose pathogen, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., is a major cause of disease in the avocado industry, causing significant economic losses, and infects all cultivars. In South Africa, Fuerte and Hass varieties are the most widely grown. Identification of genes differentially expressed in avocado during infection with the fungus represents an important step towards understanding the plant’s defence responses and would assist in designing appropriate intervention strategies. In this study, 454 sequencing and analysis of the transcriptome of infected Fuerte avocado fruits were performed using the Roche 454 GS FLX Titanium platform. cDNA libraries enriched for differentially expressed genes were constructed from unharvested and harvested avocado fruit tissues collected after 1, 4 and 24 h post-infection and after 3, 4, 5 and 7 day post-infection, then sequenced.The expression profiles of the genes expressed were measured by a hierarchical clustering algorithm.Subsequently, quantitative real-time PCR was employed to measure the expression of some candidate resistance genes to anthracnose disease and to validate the sequencing results. The single sequencing run produced 215 781 reads from the transcriptome. A total of 70.6 MB of sequence data was generated and subjected to BLAST searches of which about 1500 genes encoding proteins predicted to function in signal transduction, transcriptional control, metabolism, defence, stress response, transportation processes and some genes with unknown functions were identified. The expression profiles studies showed that many expressed genes were either up or down regulated after infection in avocado fruits when compared to the uninfected sample. Salicylic acid and ethylene were identified to be involved in the signalling networks activated in avocado fruit during C. gloeosporioides infection. This study showed that avocado is able to respond to C. gloeosporioides infection by exhibiting a sophisticated molecular system for pathogen recognition and by activating structural and biochemical defence mechanisms.

Thesis (M.Agric. (Plant Production)) -- University of Limpopo, 2018 / Meloidogyne incognita race 2 is internationally recognised as one of the most aggressive Meloidogyne species and it is also widely distributed in Limpopo Province, where it occurs alone or as mixed populations with other Meloidogyne species. Traditionally, Meloidogyne species had been managed using synthetic chemical nematicides, most of these products had been withdrawn from agro-chemical markets due to their environment-unfriendliness. Following the withdrawal of synthetic chemical nematicides, nematode resistance had been the most preferred strategy for managing high nematode population densities. The availability of nematode resistant genotypes in sweet potato (Ipomoea batatas) would enhance the use of resistance in managing Meloidogyne species and races in Limpopo Province. Generally, should post-infectional nematode resistance be available in the test sweet potato cultivars, the information would be relayed to plant breeders for use as source of introgression in various commercial cultivars where nematode-resistant genotypes do not exist. The objectives of the study, were to determine: (1) Host-status and host-sensitivity in sweet potato cv. ʹBopheloʹ, ʹBosbokʹ and ʹMvuvheloʹ to M. incognita race 2. (2) the existing nematode resistance mechanism in any of the test cultivars that had resistance to M. incognita race 2. For achieving Objective 1, eight treatments namely, 0, 25, 50, 125, 250, 625, 1250 and 3125 eggs and second stage-juveniles (J2) M. incognita race 2 were used under greenhouse trials for each cultivar. To achieve Objective 2, sweet potato plants were inoculated with 100 J2 with four plants harvested every other day for 30 days counting to 15 harvesting times. At 56 days after inoculation, cv. ʹBopheloʹ had reproductive factor (RF) values above unity for M. incognita race 2 and plant growth variables were reduced. Therefore, the cultivar was a susceptible host to M. incognita race 2 and mechanism trial was not conducted for this cultivar. Meloidogyne incognita race 2 failed to reproduce on cultivars ʹBosbokʹ and ʹMvuvheloʹ whereas nematode infection did not affect plant growth and therefore, the two cultivars were resistant to M. incognita race 2. Mechanisms of resistance to M. incognita race 2 on cultivars ʹBosbokʹ and ʹMvuvheloʹ demonstrated significance existence of (1) necrotic spots, (2) poorly developed giant cells, (3) formation of rootlet interferences (4) absence of root galls and (5) non-detectable J2 in roots. All these features suggested the existence of post-infectional nematode resistance in the two cultivars to M. incognita race 2. In conclusion, cultivar ʹBopheloʹ was susceptible to M. incognita race 2, whereas cultivars ʹBosbokʹ and ʹMvuvheloʹ were resistant to M. incognita race 2, with the evidence of post-infectional nematode resistance to the nematode species

Thesis (Ph.D. (Medical Science)) -- University of Limpopo, 2012

No description available.

This work was aimed to investigate the basic viral infection protocols mainly focusing on Nicotiana benthamiana hairy root cultures and wild-type tobacco mosaic virus (TMV). The application of transgenic virus containing the gene for green fluorescent protein (GFP) for foreign protein production in plant tissue cultures and whole plants was also studied. The effect on viral accumulation of the form of plant tissue culture used, such as hairy roots, shooty teratomas and suspended cells, was investigated. Viral infection was shown to have no effect on culture growth and morphology. Hairy root cultures are a superior host for viral propagation and production in vitro. The maximum specific rate of viral accumulation occurred mainly during the root growth phase. The average maximum virus concentration in the hairy roots was 0.82 ?? 0.14 mg g-1 dry weight and virus protein represented a maximum of approximately 6% of total soluble protein in the root biomass. Proportional scale-up of TMVinfected hairy roots in shake flasks and bioreactors can be achieved without changing the average virus concentration accumulated in the hairy roots. The level of viral accumulation was much lower in N. benthamiana hairy roots infected with transgenic virus containing GFP (TMVGFPC3) compared with TMV and low levels or no GFP was detected. Viral accumulation and GFP production in whole plants was studied using different generations of transgenic TMV-GFPC3 virus. Hybrid viruses with the foreign gene GFPC3 deleted may have been formed in successive TMV-GFPC3 generations, resulting in the loss of GFP production and enhanced viral infectivity. In vitro generated RNA transcript and first generation TMV-GFPC3 were found to be more suitable for infection than the second generation TMV-GFPC3. However, the accumulation of GFP and virus concentration did not occur at the same ratio. Provided a more genetically stable transgenic viral vector is used for infection, transient viral infection of hairy roots can be a potential alternative system for foreign protein production than plants grown in the field as the containment or safety issues can be addressed.

Includes bibliographical references (leaves 241-25). Aims to evaluate sampling procedures; assess the extent and magnitude of yield loss caused by Pratylenchus neglectus; assess the population dynamics of Pratylenchus neglectus in cereals; determine whether resistance occurs in field crops; and, assess whether variation occurs between geographically isolated species of Pratylenchus neglectus