Characterization of Citrus tristeza virus isolates from grapefruit (Citrus paradisi Macf.) accessions of Citrus Active Germplasm Bank

Citrus tristeza virus (CTV) isolates from 35 grapefruit accessions belonging to Citrus Active Germplasm Bank of the "Instituto Agronômico de Campinas" located at the "Centro APTA Citros Sylvio Moreira", Cordeirópolis, São Paulo state, Brazil, were characterized and evaluated through symptoms in the trees, biological indexing, immunological diagnosis with different monoclonal antibodies and SSCP analysis (single-strand conformation polymorphism) of the coat protein gene. Symptomatology indicated that, in general, the group of plants with smaller canopy volume and severe stem pitting differed significantly from the group that presented greater vegetative development and mild to moderate stem pitting. However, the isolates from most of the accessions induced mild reaction on Mexican lime. The serological evaluation through the DAS-ELISA using monoclonal antibodies did not reveal any association between virus titer in the plant tissue and symptoms. The reaction with different monoclonal antibodies and the distinct electrophoresis patterns obtained through SSCP showed that there is a high degree of diversity among the isolates that infect these grapefruit accessions. High complexity within the same isolate was also observed in the SSCP profiles. This finding indicates that the CTV isolates from these plants are a complex mixture of CTV haplotypes. Similar SSCP banding patterns were observed among some plants with strong stem pitting symptoms, and among some plants with weak or moderate stem pitting symptoms.

Agressividade de linhagens de Xanthomonas axonopodis pv. aurantifolii Tipo C em lima ácida 'Galego'

O presente trabalho objetivou gerar informações referentes à agressividade de linhagens de Xanthomonas axonopodis pv. aurantifolii Tipo C(Xaa-C), produtoras (PP) e não produtoras de pigmento (NP) escuro em meio de cultura, comparativamente a X. axonopodis pv. citri Tipo A (Xac) . Os tratamentos foram formados por 14 linhagens, sendo sete de Xaa-C PP, cinco Xaa-C NP, e duas linhagens de Xac. As linhagens foram inoculadas através de ferimentos, em folhas de lima ácida 'Galego' (Citrus aurantifolia Swingle), com agulha previamente mergulhada em uma suspensão de células bacterianas (10(7) UFC/mL). Foram realizadas dez repetições para cada tratamento, representadas por uma planta cada. As plantas foram mantidas em casa de vegetação durante todo o experimento. As linhagens diferiram entre si quanto ao período de incubação, diâmetro e populações bacterianas das lesões e, comparativamente, Xaa-C NP mostraram-se mais agressivas do que Xaa-C PP. Algumas linhagens induziram sintomas que diferiram quanto à presença e extensão de anasarca, halo amarelo e saliência do tecido necrosado.

Expressão gênica diferencial de laranja Pêra Rio (Citrus sinensis (L.) Osbeck) e Lima Ácida 'Galego' (Citrus aurantifolia Swingle) em resposta à infecção por Xanthomonas citri subsp. citri

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Expressão gênica de Xanthomonas citri subsp. citri colonizando laranja doce ‘pêra rio’ (Citrus sinensis (L.) Osbeck) e lima ácida ‘galego’ (Citrus aurantifolia Swingle)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the non-inoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range

Abstract Background Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain Aw12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime. Results To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306. Conclusions Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

Mecanismos implicados en las etapas iniciales de infección en la cancrosis de los cítricos provocada por Xanthomonas citri subsp. citri

La cancrosis o chancro bacteriano de los cítricos (CBC) causada por Xanthomonas citri subsp. citri (Xcc) y X. fuscans subsp. aurantifolii, afecta a un gran número de especies dentro de la familia de las rutáceas, especialmente cítricos. Esta enfermedad produce graves pérdidas económicas allí donde está presente, principalmente porque la comercialización de cítricos desde las zonas afectadas hacía zonas libres de cancrosis, está sujeta a fuertes medidas cuarentenarias. La cancrosis se encuentra distribuida a nivel mundial pero no se ha localizado ni en la Unión Europea ni en ningún área del Mediterráneo. Se han descrito tres tipos de cancrosis en función de la gama de huésped y de las características fenotípicas y genotípicas de las bacterias que las producen. La más extendida es la cancrosis tipo A producida por Xcc, dentro de la cual se distinguen los subtipos Aw y A*, originarios de Florida y Sudeste Asiático, respectivamente, que de forma natural solo son capaces de producir enfermedad en lima mejicana. En este trabajo se presentan estudios sobre mecanismos implicados en las primeras etapas de la infección, como la quimiotaxis y formación de biopelículas, en la cancrosis de los cítricos. La quimiotaxis es el proceso por el cual las bacterias se dirigen hacia zonas favorables para su supervivencia y desarrollo. Los perfiles quimiotácticos obtenidos frente a distintas fuentes de carbono, así como los estudios en relación al contenido de proteínas aceptoras de grupos metilo (MCPs), permitieron agrupar a las cepas de Xanthomonas estudiadas en este trabajo, de acuerdo a la enfermedad producida y a su gama de huésped. Todas las cepas mostraron quimiotaxis positiva frente a extractos de hoja y apoplasto de diferentes especies, sin embargo, Xcc 306, X. alfalfae subsp. citrumelonis (Xac) y X. campestris pv. campestris (Xc) manifestaron respuestas más específicas frente a extractos de apoplasto de hojas de naranjo dulce, lima y col china, respectivamente. Dicho resultado nos permite asociar el mecanismo de quimiotaxis con la capacidad de las cepas de Xanthomonas para colonizar estos huéspedes de forma específica. Las cepas estudiadas fueron capaces de realizar movimiento tipo swimming, twitching y sliding en distintos medios, siendo el movimiento swimming el único en el que se encontraron diferencias entre las cepas de Xcc con distinta gama de huésped. En este trabajo se ha estudiado además la formación de biopelículas en superficies bióticas y abióticas, un mecanismo importante tanto para la supervivencia en superficie vegetal como para el desarrollo de la infección. Las cepas de Xanthomonas estudiadas fueron capaces de formar biopelículas in vitro, siendo mayor en un medio que simula el apoplasto y que contiene una baja concentración de nutrientes en comparación con medios que contenían alta concentración de nutrientes. La formación de biopelículas en superficie vegetal se encontró relacionada, en las cepas patógenas de cítricos, con la capacidad para infectar un tejido o huésped determinado. Se han caracterizado algunos de los componentes de la matriz extracelular producida por Xcc, que compone hasta un 90% de las bipoelículas. Entre ellos destaca el ADN extracelular, que tiene un papel como adhesina en las primeras etapas de formación de biopelículas y estructural en biopelículas maduras. Además, se han identificado el pilus tipo IV como componente importante en las biopelículas, que también participa en motilidad. Finalmente, se han realizado estudios sobre la expresión de genes implicados en motilidad bacteriana y formación de biopelículas que han confirmado las diferencias existentes entre cepas de Xcc de amplia y limitada gama de huésped, así como el papel que juegan elementos como el pilus tipo IV o el flagelo en estos procesos. ABSTRACT Xanthomonas citri subsp. citri (Xcc) and X. fuscans subsp. aurantifolii are the causal agents of Citrus Bacterial Canker (CBC) which is one of the most important citrus diseases. CBC affects all Citrus species as well as other species from Rutaceae family. CBC produces strong economic losses; furthermore the commercialization of plants and fruits is restricted from infested to citrus canker free areas. The disease is worldwide distributed in tropical and subtropical areas, however it is not present in the European Union. Three types of CBC have been described according to the host range and phenotypic and genotypic characteristics. CBC type A caused by Xcc is he widest distributed. Within CBC A type two subtypes Aw and A* were described from Florida and Iran respectively, both infecting only Mexican lime. Herein mechanisms connected to early events in the citrus bacterial canker disease such as chemotaxis and biofilm formation, were studied. Chemotaxis allows bacteria to move towards the more suitable environments for its survival, host colonization and infection. Studies performed on citrus pathogenic Xanthomonas and X. campestris pv. campestris (Xc), a crucifer pathogen, have shown different chemotactic profiles towards carbon compound as well as different MCPs profile, which clustered strains according to host range and disease caused. Every strain showed positive chemotaxis toward leaf extracts and apoplastic fluids from sweet orange, Mexican lime and Chinese cabbage leaves. However, a more specific response was found for strains Xcc 306, X. alfalfae subsp. citrumelonis and Xc towards sweet orange, Mexican lime and Chinese cabbage apoplastic fluids, respectively. These results relate chemotaxis with the higher ability of those strains to specifically colonize their proper host. Xanthomonas strains studied were able to perform swimming, sliding and twitching motilities. The ability to swim was variable among CBC strains and seemed related to host range. Biofilm formation is an important virulence factor for Xcc because it allows a better survival onto the plant surface as well as facilitates the infection process. The studied Xanthomonas strains were able to form biofilm in vitro, on both nutrient rich and apoplast mimicking media, furthermore the biofilm formation by all the strains was higher in the apoplast mimicking media. The ability to form biofilm in planta by Xcc and Xac strains was dependent of the host and the tissue colonized. The wide host range CBC strain was able to form biofilm onto several citrus leaves and fruits, however the limited host range CBC strain produced biofilm solely onto Mexican lime leaves and fruits. Furthermore Xac strain, which solely infects leaves of young plants, was not able to develop biofilms on fruits. Some components of the extracellular matrix produced by Xcc strains have been characterized. Extracellular DNA acted as an adhesin at the very early stages of biofilm formation and as structural component of mature biofilm for citrus pathogenic Xanthomonas. Furthermore type IV pilus has been identified as a component of the extracellular matrix in biofilm and motility. Transcriptional studies of genes related with biofilm formation and motility have confirmed the differential behavior found among wide and limited host range CBC strains as well as the role of type IV pili and flagellum on those processes.

Macronutrients in Marandu Palisade Grass as Influenced by Lime, Slag, and Nitrogen Fertilization

Soil acidity is one of the main limiting factors for the growth of pasture grasses in Brazilian soils. In addition to lime, slag can be used to correct soil acidity and help plants to absorb nutrients in adequate amounts. The objective of this experiment was to evaluate, under greenhouse conditions, the effects of slag and lime plus nitrogen (N) on marandu palisade grass plants` nutritional status as well as the absorption of macronutrients submitted to two cuts. The treatments consisted of two corrective materials (slag and lime), three corrective material rates (0.81, 1.61, and 3.22 g dm-3 of ECaCO3), three N rates (75, 150, and 300 mg dm-3) plus a control treatment, with four replications. Macronutrient contents in the forage plants were found to be present in adequate levels. The mean value of N accumulated in the shoot was 40.1 mg per plant, phosphorus (P) was 4.6 mg per plant, potassium (K) was 38.6 mg per plant, calcium (Ca) was 7.3 mg per plant, magnesium (Mg) was 6.7 mg per plant, and sulfur (S) was 3.5 mg per plant at the first cut. At the second cut, the nutrient accumulations values were N 50.8 mg per plant, P 6.3 mg per plant, K 20.7 mg per plant, Ca 21.6 mg per plant, Mg 24.0 mg per plant, and S 4.7 mg per plant. Macronutrients accumulation in the shoot of grass increased with the addition of both the correctives as well as the N rates.

Mechanical properties, drying and autogenous shrinkage of blast furnace slag activated with hydrated lime and gypsum

This article reports the characteristics of blast furnace slag (BFS) pastes activated with hydrated lime (5%) and hydrated lime (2%) plus gypsum (6%) in relation to compressive strength, shrinkage (autogenous and drying) and microstructure (porosity, hydrated products). The paste mixtures were characterized using powder X-ray diffraction (XRD), mercury intrusion porosimetry (MIP) and thermogravimetric analysis (TG/DTG). BSF activated with lime and gypsum (LG) results in larger amounts of ettringite when compared with BFS activated with lime (L). Although the porosities of the L and LG mixtures were about the same, there was a greater pore refinement for the BFS activated with lime, with an increase in mesopores volume with age. The presence of ettringite and the higher volumes of macropores cause the compressive strength of BSF activated with hydrated lime plus gypsum to be smaller than that of BFS activated with lime. For both chemical activators, compressive strength developed slowly at early ages. Autogenous and drying shrinkage were greater for the BFS activated with lime, believed to result from the more refined porous structure in comparison with the mixture activated with gypsum plus lime. (c) 2010 Elsevier Ltd. All rights reserved.

Carbide lime and industrial hydrated lime characterization

This paper focuses on the characterization of carbide lime (CL) - a by-product of acetylene production, composed mainly of calcium hydroxide with minor parts of carbonate - and compares its features to those of ""dry"" hydrated lime (HL) commonly used as a building material. Chemical, thermogravimetric and X-ray diffraction analyses indicated that the limes are similar in chemical and mineralogical compositions. except for the presence of carbon in the waste. Morphological and elemental chemical analyses by SEM and EDS revealed that CL particles differ from HL ones in their morphology and by the presence of carbon formations, Physical characterization included density and BET surface area of the materials. as well as, their particle size distributions in deionized water at diverse time periods. CL underwent agglomeration after approximately 60 min in water, whereas HL progressively became finer with time as determined by laser diffraction. In addition, water retention and squeeze flow tests were used to assess the pastes` fresh properties. (c) 2009 Elsevier B.V. All rights reserved.

Modelling the progress of Asiatic citrus canker on Tahiti lime in relation to temperature and leaf wetness

The combined effect of temperature (15A degrees C, 20A degrees C, 25A degrees C, 30A degrees C, 35A degrees C, 40A degrees C and 42A degrees C) and leaf wetness duration (0, 4, 8 12, 16, 20 and 24 h) on infection and development of Asiatic citrus canker (Xanthomonas citri subsp. citri) on Tahiti lime plant was examined in growth chambers. No disease developed at 42A degrees C and zero hours of leaf wetness. Periods of leaf wetness as short as 4 h were sufficient for citrus canker infection. However, a longer leaf duration wetness (24 h) did not result in much increase in the incidence of citrus canker, but led to twice the number of lesions and four times the disease severity. Temperature was the greatest factor influencing disease development. At optimum temperatures (25-35A degrees C), there was 100% disease incidence. Maximum disease development was observed at 30-35A degrees C, with up to a 12-fold increase in lesion density, a 10-fold increase in lesion size and a 60-fold increase in disease severity.

Performance of `Tahiti` lime on twelve rootstocks under irrigated and non-irrigated conditions

Faced with new challenges, such as emerging diseases, shortening of orchard longevity, and larger social and environmental demands from consumers, practices such as rootstock diversification, irrigation and high density plantings have become relevant for the Brazilian citrus industry. This research had the objective to evaluate the performance of irrigated and non-irrigated `Tahiti` lime trees grafted on 12 rootstocks and one interstock. Plots were distributed following a randomized block design, with four replicates and one plant per plot. Rootstocks influenced plant vigor, especially `Flying Dragon` trifoliate, which reduced tree height by approximately 47% compared to the `Rangpur lime. Trees that were budded on more vigorous rootstocks showed higher yield when grown without irrigation than with irrigation. The `1646` citradia and `Morton` citrange rootstocks performed particularly well. On the other hand, the plants on less vigorous rootstocks showed better performance in terms of yield under irrigation than the same combinations without irrigation, especially those grafted on the tetraploid `Carrizo` and `Troyer` citranges, `Swingle` citrumelo, `Davis A` trifoliate and `Flying Dragon` trifoliate. Plants budded on the `1708` citradia had high yields under irrigated and non-irrigated conditions. The effect of interstock on plant vigor was dependent of rootstock. Interstocked plants on `Davis A` trifoliate were higher than those without interstock. On the other hand, interstocked plants on Catania 2 `Volkamer` lemon were less vigorous than those without interstock. (C) 2011 Elsevier B.V. All rights reserved.

Viroid species associated with the bark-cracking phenotype of `Tahiti` acid lime in the State of Sao Paulo, Brazil

Viroids have been used as ""graft transmissible dwarfing agents"" (GTDA) in several countries, mainly to reduce growth of citrus trees, thus increasing their density in orchards. In the State of Sao Paulo, Brazil, plants of the acid lime `Tahiti` are usually grafted with a complex of GTDA, presumably viroids. The aim of the present work was the identification and molecular characterization of the viroids infecting trees of acid lime `Tahiti` displaying ""Quebra galho"" (bark-cracking). Viroids were identified and characterized by biological indexing in `Etrog` citron, Northern-blot hybridization, RT-PCR, cloning and complete sequencing of the RNA genomes. Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus dwarfing viroid (CDVd) were found in different combinations. Although we have not been able to infer a direct relationship between the agronomical performance and symptom severity with the presence of a specific viroid or viroid combination, the differences in the severity of ""Quebra-galho"" symptoms among different trees is probably associated with the presence (or absence) of CEVd, with its interaction with other viroids perhaps determining the different phenotypes observed in the field.