RESUMO
Huanglongbing (HLB) is currently the most devastating disease of citrus worldwide. Both bacteria 'Candidatus Liberibacter asiaticus' (CLas) and 'Candidatus Liberibacter americanus' (CLam) are associated with HLB in Brazil but with a strong prevalence of CLas over CLam. Conventionally, HLB management focuses on controlling the insect vector population (Diaphorina citri; also known as Asian citrus psyllid [ACP]) by spraying insecticides, an approach demonstrated to be mostly ineffective. Thus, development of novel, more efficient HLB control strategies is required. The multifunctional bacterial outer membrane protein OmpA is involved in several molecular processes between bacteria and their hosts and has been suggested as a target for bacterial control. Curiously, OmpA is absent in CLam in comparison with CLas, suggesting a possible role in host interaction. Therefore, in the current study, we have treated ACPs with different OmpA-derived peptides, aiming to evaluate acquisition of CLas by the insect vector. Treatment of psyllids with 5 µM of Pep1, Pep3, Pep5, and Pep6 in artificial diet significantly reduced the acquisition of CLas, whereas increasing the concentration of Pep5 and Pep6 to 50 µM abolished this process. In addition, in planta treatment with 50 µM of Pep6 also significantly decreased the acquisition of CLas, and sweet orange plants stably absorbed and maintained this peptide for as long as 3 months post the final application. Together, our results demonstrate the promising use of OmpA-derived peptides as a novel biotechnological tool to control CLas.
Assuntos
Citrus , Hemípteros , Rhizobiaceae , Animais , Liberibacter , Peptídeos , Doenças das PlantasRESUMO
The bacteria Xylella fastidiosa is the causative agent of a number of economically important crop diseases, including citrus variegated chlorosis. Although its complete genome is already sequenced, X. fastidiosa is very poorly characterized by biochemical approaches at the protein level. In an initial effort to characterize protein expression in X. fastidiosa we used one- and two-dimensional gel electrophoresis and mass spectrometry to identify the products of 142 genes present in a whole cell extract and in an extracellular fraction of the citrus isolated strain 9a5c. Of particular interest for the study of pathogenesis are adhesion and secreted proteins. Homologs to proteins from three different adhesion systems (type IV fimbriae, mrk pili and hsf surface fibrils) were found to be coexpressed, the last two being detected only as multimeric complexes in the high molecular weight region of one-dimensional electrophoresis gels. Using a procedure to extract secreted proteins as well as proteins weakly attached to the cell surface we identified 30 different proteins including toxins, adhesion related proteins, antioxidant enzymes, different types of proteases and 16 hypothetical proteins. These data suggest that the intercellular space of X. fastidiosa colonies is a multifunctional microenvironment containing proteins related to in vivo bacterial survival and pathogenesis. A codon usage analysis of the most expressed proteins from the whole cell extract revealed a low biased distribution, which we propose is related to the slow growing nature of X. fastidiosa. A database of the X. fastidiosa proteome was developed and can be accessed via the internet (URL: www.proteome.ibi.unicamp.br).
Assuntos
Proteínas de Bactérias/química , Gammaproteobacteria/metabolismo , Proteoma , Antioxidantes/química , Aderência Bacteriana , Códon , Bases de Dados como Assunto , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Gammaproteobacteria/química , Genoma Bacteriano , Processamento de Imagem Assistida por Computador , Ferro/metabolismo , Espectrometria de Massas , Fases de Leitura Aberta , Peptídeos/química , Doenças das Plantas/microbiologia , Porinas/fisiologiaRESUMO
Xylella fastidiosa was isolated from sweet orange plants (Citrus sinensis) grown in two orchards in the northwest region of the Brazilian state of São Paulo. One orchard was part of a germ plasm field plot used for studies of citrus variegated chlorosis resistance, while the other was an orchard of C. sinensis cv. Pêra clones. These two collections of strains were genotypically characterized by using random amplified polymorphic DNA (RAPD) and variable number of tandem repeat (VNTR) markers. The genetic diversity (H(T)) values of X. fastidiosa were similar for both sets of strains; however, H(T)(RAPD) values were substantially lower than H(T)(VNTR) values. The analysis of six strains per plant allowed us to identify up to three RAPD and five VNTR multilocus haplotypes colonizing one plant. Molecular analysis of variance was used to determine the extent to which population structure explained the genetic variation observed. The genetic variation observed in the X. fastidiosa strains was not related to or dependent on the different sweet orange varieties from which they had been obtained. A significant amount of the observed genetic variation could be explained by the variation between strains from different plants within the orchards and by the variation between strains within each plant. It appears, therefore, that the existence of different sweet orange varieties does not play a role in the population structure of X. fastidiosa. The consequences of these results for the management of sweet orange breeding strategies for citrus variegate chlorosis resistance are also discussed.
Assuntos
Citrus/classificação , Citrus/microbiologia , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Doenças das Plantas/microbiologia , Técnicas de Tipagem Bacteriana , DNA Bacteriano/análise , Variação Genética , Genótipo , Repetições Minissatélites/genética , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
In citrus improvement programs the characterization of the available genetic resources is of great importance, mainly concerning biological characteristics of the culture, as the heterozigosity, nucellar the embriony and long reproductive cycle. Favored by nucellar embriony interespecific and intergeneric hybridizations and genotypes preservation happen easily. RAPDs markers were used to analyze 15 Citrus spp., four sweet orange (C. sinensis Osbeck), (C. medica, C. grandis e C. latifolia), four mandarins (C. reticulata Blanco, C. nobilis Loureiro, C. sunki Loureiro e C. deliciosa Tenore), a sour orange (C. aurantium L.), a grapefruit (C. paradisi Marcf.), a pummelo (C. grandis Osbeck), a cidra (C. medica L.), a lime (C. latifolia) and two hybrids (Citrus clementina T. x (C. tangerina T. x C. paradisi Macf.)). Genetic similarities of 15 Citrus genotypes obtained with twelve random primers, indicated a minimum similarity degree of 0.81 (simple matching) among the mandarins. Lower similarity degrees were obtained among less related Citrus species (C. medica, C. grandis e C. latifolia). The four varieties of sweet oranges (C. sinensis Osbeck) could not be differentiated by RAPD markers, showing maximum similarity.
Em programas de melhoramento de citros, a caracterização adequada dos recursos genéticos disponíveis é de grande importância, principalmente devido às características biológicas da cultura, como a heterozigosidade, a embrionia nucelar e o longo ciclo reprodutivo. A facilidade com que ocorrem hibridações (interespecíficas e intergenéricas) e a embrionia nucelar favoreceram a formação e a preservação de novas combinações, classificadas como espécies. Neste estudo, marcadores RAPDs foram utilizados para analisar 15 acessos de Citrus spp., sendo quatro variedades de laranjeiras doce (C. sinensis Osbeck), quatro tangerineiras (C. reticulata Blanco, C. nobilis Loureiro, C. sunki Loureiro e C. deliciosa Tenore), uma laranjeira azeda (C. aurantium L.), um pomeleiro (C. paradisi Macf.), uma torangeira (C. grandis Osbeck), uma cidreira (C. medica L.), uma limeira ácida (C. latifolia) e dois híbridos (Citrus clementina T. x (C. tangerina T. x C. paradisi Macf.)). Doze sequências iniciadoras aleatórias foram utilizadas para estudar os 15 genótipos, encontrando-se um grau de similaridade mínimio de 0,81 ("Simple Matching") entre as tangerineiras. Os menores graus de similaridade foram encontrados entre as espécies de Citrus menos aparentadas (C. medica, C. grandis e C. latifolia). As quatro cultivares de laranjeiras doces não puderam ser diferenciadas pelos marcadores RAPD utilizados, apresentando similaridade máxima.
RESUMO
In citrus improvement programs the characterization of the available genetic resources is of great importance, mainly concerning biological characteristics of the culture, as the heterozigosity, nucellar the embriony and long reproductive cycle. Favored by nucellar embriony interespecific and intergeneric hybridizations and genotypes preservation happen easily. RAPDs markers were used to analyze 15 Citrus spp., four sweet orange (C. sinensis Osbeck), (C. medica, C. grandis e C. latifolia), four mandarins (C. reticulata Blanco, C. nobilis Loureiro, C. sunki Loureiro e C. deliciosa Tenore), a sour orange (C. aurantium L.), a grapefruit (C. paradisi Marcf.), a pummelo (C. grandis Osbeck), a cidra (C. medica L.), a lime (C. latifolia) and two hybrids (Citrus clementina T. x (C. tangerina T. x C. paradisi Macf.)). Genetic similarities of 15 Citrus genotypes obtained with twelve random primers, indicated a minimum similarity degree of 0.81 (simple matching) among the mandarins. Lower similarity degrees were obtained among less related Citrus species (C. medica, C. grandis e C. latifolia). The four varieties of sweet oranges (C. sinensis Osbeck) could not be differentiated by RAPD markers, showing maximum similarity.
Em programas de melhoramento de citros, a caracterização adequada dos recursos genéticos disponíveis é de grande importância, principalmente devido às características biológicas da cultura, como a heterozigosidade, a embrionia nucelar e o longo ciclo reprodutivo. A facilidade com que ocorrem hibridações (interespecíficas e intergenéricas) e a embrionia nucelar favoreceram a formação e a preservação de novas combinações, classificadas como espécies. Neste estudo, marcadores RAPDs foram utilizados para analisar 15 acessos de Citrus spp., sendo quatro variedades de laranjeiras doce (C. sinensis Osbeck), quatro tangerineiras (C. reticulata Blanco, C. nobilis Loureiro, C. sunki Loureiro e C. deliciosa Tenore), uma laranjeira azeda (C. aurantium L.), um pomeleiro (C. paradisi Macf.), uma torangeira (C. grandis Osbeck), uma cidreira (C. medica L.), uma limeira ácida (C. latifolia) e dois híbridos (Citrus clementina T. x (C. tangerina T. x C. paradisi Macf.)). Doze sequências iniciadoras aleatórias foram utilizadas para estudar os 15 genótipos, encontrando-se um grau de similaridade mínimio de 0,81 ("Simple Matching") entre as tangerineiras. Os menores graus de similaridade foram encontrados entre as espécies de Citrus menos aparentadas (C. medica, C. grandis e C. latifolia). As quatro cultivares de laranjeiras doces não puderam ser diferenciadas pelos marcadores RAPD utilizados, apresentando similaridade máxima.