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Master's Dissertation
DOI
10.11606/D.11.2008.tde-25072008-104142
Document
Author
Full name
João Paulo Rodrigues Marques
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2008
Supervisor
Committee
Gloria, Beatriz Appezzato da (President)
Fernando, Juliana Aparecida
Tanaka, Francisco Andre Ossamu
Title in Portuguese
Análise histopatológica e perfil protéico de lesões causadas pelo Citrus leprosis virus tipo nuclear e citoplasmático em Citrus sinensis (l.) Osbeck
Keywords in Portuguese
Anatomia vegetal
Fitopatologia
Frutas cítricas
Leprose.
Abstract in Portuguese
A leprose dos citros é uma doença de grande importância econômica aos citricultores, devido aos prejuízos por ela causados. A doença provoca lesões localizadas nos ramos, frutos e folhas e pode causar grandes perdas aos pomares não tratados. A leprose dos citros possui etiologia viral sendo o vírus denominado Citrus leprosis virus (CiLV). Atualmente, atribuí-se a leprose dois tipos de vírus que causam sintomas morfológicos e citopatológicos distintos: o tipo nuclear (CiLV-N) e o tipo citoplasmático (CiLV-C). O presente trabalho teve como objetivo investigar as alterações anatômicas e comparar o perfil protéico entre as lesões causadas pelos dois tipos de vírus em laranjeira-doce [Citrus sinensis (L.) Osbeck]. Para as análises anatômicas foram coletadas amostras de tecidos sadios e de tecidos lesionados. As amostras foram fotografadas e em seguida fixadas em solução de Karnovsky, desidratadas em série etílica, infiltradas em resina hidroxi-etil-metacrilato (Leica Historesin), seccionadas em micrótomo rotativo a 5-7 µm de espessura, coradas com azul de Toluidina para as análises histológicas usuais. Também foram realizados testes histoquímicos para fenóis, compostos pécticos, proteína e lipídios. A captura de imagens digitais dos materiais preparados em lâminas foi realizada em microscópio equipado com câmera de vídeo. Para a análise do perfil protéico as amostras coletadas foram imediatamente criofixadas em nitrogênio líquido e armazenadas em refrigerador a - 80ºC para posterior extração protéica. As proteínas extraídas foram quantificadas e depois separadas por eletroforese em gel de policrilamida. As lesões foliares se iniciaram como pontuações necróticas, envolvidas por halos cloróticos que limitam o crescimento da lesão. Nos ramos, ocorreram dois tipos de lesões com fendas. As lesões de ramos e de folhas provenientes do campo com sintomas de CiLV-C apresentaram similaridades com as amostras inoculadas. No caso das lesões foliares causadas pelo CiLV-N observa-se a presença de três regiões distintas: o centro necrótico, o halo intermediário e o halo clorótico. As lesões nos frutos de ambos os tipos de leprose eram distintas. Ductos traumáticos gomosos foram observados nos ramos infectados pelo CiLV-C e nos feixes vasculares de folhas e frutos infectados pelo CiLV-N. Quanto ao perfil protéico, verificou-se um aumento na concentração protéica nos tecidos infectados, em especial, nas lesões causadas pelo CiLV-N. Foi possível observar que havia um padrão anatômico e histoquímico nas lesões causadas pela leprose em laranjeira doce independente do tipo de vírus, pois em todas as lesões foi verificada hipertrofia e hiperplasia celular, plasmólise, a redução de grãos de amido, o acúmulo de compostos lipídicos nas células da área necrosada e a presença de um halo, ao redor dessa área, constituído de células que acumulam proteínas.
Title in English
Histopathological analysis and proteic profile of lesions caused by Citrus leprosis virus nuclear and cytoplasmatic type in Citrus sinensis (L.) Osbeck
Keywords in English
Citrus leprosis
Citrus sinensis.
Histopathology
Abstract in English
The citrus leprosis of citrus has been considered a disease of great economic importance to citrus producers due to the damage that it causes. The disease is responsible for lesions found in the twigs, leaves and fruits, and it may cause great losses to the untreated orchards. The citrus leprosis has viral etiology being the virus called Citrus leprosis virus (CiLV). Currently, there are two types of virus attributed to leprosis that cause different morphological and cythopatological symptoms: the nuclear (CiLV-N) and cytoplasmic type (CiLV-C). This study aimed to investigate the anatomical changes and to compare the proteic profile between the injuries caused by two types of virus in sweet orange [Citrus sinensis (L.) Osbeck]. For anatomical analyses healthy tissue and different lesions were photographed and then fixed in Karnovsky solution, dehydrated in a graded ethylic series, embedded in hidroxy-ethyl methacrylate resin (Leica Historesin), sectioned (5 ?m thick), stained and mounted in synthetic resin. The digital images were capture in a microscope with video camera. For the analysis of protein profiles the samples were collected and immediately cryofixed in liquid nitrogen and stored in the refrigerated - 80 ° C. The proteins were extracted, quantified and then separated by gel electrophoresis of polyacrylamide gel. Foliar lesions began as necrotic spots surrounded by a yellow halo that inhibited the lesion expansion. The twigs presented two different lesions: depressed ones and pustules with ruptures. The collected-field lesions of leaves and twigs showed that symptoms of CiLV-C had a certain similarity with the inoculated samples. In the case of foliar lesions caused by CiLV-N there was the presence of three different regions: the necrotic center, the intermediate halo and chlorotic halo. The fruit lesions caused by both types of leprosis virus were different. Ductos traumáticos gomosos foram observados nos ramos infectados pelo CiLV-C e nos feixes vasculares de folhas e frutos infectados pelo CiLV-N. Gummous traumatic ducts in twigs infected by CiLV-C and in the vascular bundles of fruit and leaves infected with CiLV-N were observed. In the proteic profile, there was an increase in protein concentration in the infected tissues especially for injuries caused by CiLV-N. It was possible to observe that there was an anatomical and histochemical pattern in the lesions caused by leprosis in sweet orange independently of the type of virus, therefore in all the injuries was verified hyperplasia, hypertrophy, cell plasmolisis, the reduction of starch grains, the accumulation of lipids compounds in the cells of the necrotic areas and the presence of a halo, surrounding that area, constituted by cells that accumulate proteins.
 
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Publishing Date
2008-07-29
 
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