Sistema para depleção de proteínas em Xanthomonas citri subsp. citri

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Greenhouse gas balance associated with sugarcane production in South-Central Brazil, considering the management and expansion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Seleção de sítio de divisão em Xanthomonas citri subsp. citri: caracterização de minC.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Caracterização funcional do mutante da ORF XAC1008 de Xanthomonas citri subsp. citri (Xac)

Pós-graduação em Microbiologia Agropecuária - FCAV

On the effect of aromatherapy with citrus fragrance in the therapy of major depressive disorder

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mutagênese sítio-dirigida da ORF XAC0024 de Xanthomonas citri subsp. citri e suas implicações no desenvolvimento do cancro cítrico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Accuracy, precision and robustness of different methods to obtain samples from silages in fermentation studies

The objective of this study was to evaluate accuracy, precision and robustness of two methods to obtain silage samples, in comparison with extraction of liquor by manual screw-press. Wet brewery residue alone or combined with soybean hulls and citrus pulp were ensiled in laboratory silos. Liquor was extracted by a manual screw-press and a 2-mL aliquot was fixed with 0.4 mL formic acid. Two 10-g silage samples from each silo were diluted in 20 mL deionized water or 17% formic acid solution (alternative methods). Aliquots obtained by the three methods were used to determine the silage contents of fermentation end-products. The accuracy of the alternative methods was evaluated by comparing mean bias of estimates obtained by manual screw-press and by alternative methods, whereas precision was assessed by the root mean square prediction error and the residual error. Robustness was determined by studying the interaction between bias and chemical components, pH, in vitro dry matter digestibility (IVDMD) and buffer capacity. The 17% formic acid method was more accurate for estimating acetic, butyric and lactic acids, although it resulted in low overestimates of propionic acid and underestimates of ethanol. The deionized water method overestimated acetic and propionic acids and slightly underestimated ethanol. The 17% formic acid method was more precise than deionized water for estimating all organic acids and ethanol. The robustness of each method with respect to variation in the silage chemical composition, IVDMD and pH is dependent on the fermentation end-product at evaluation. The robustness of the alternative methods seems to be critical at the determination of lactic acid and ethanol contents.

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.

Cloning of a Tobacco Apoplasmic Invertase Inhibitor1 : Proof of Function of the Recombinant Protein and Expression Analysis during Plant Development

Higher plants express several isoforms of vacuolar and cell wall invertases (CWI), some of which are inactivated by inhibitory proteins at certain stages of plant development. We have purified an apoplasmic inhibitor (INH) of tobacco (Nicotiana tabacum) CWI to homogeneity. Based on sequences from tryptic fragments, we have isolated a full-length INH-encoding cDNA clone (Nt-inh1) via a reverse transcriptase-polymerase chain reaction. Southern-blot analysis revealed that INH is encoded by a single- or low-copy gene. Comparison with expressed sequence tag clones from Arabidopsis thaliana and Citrus unshiu indicated the presence of Nt-inh1-related proteins in other plants. The recombinant Nt-inh1-encoded protein inhibits CWI from tobacco and Chenopodium rubrum suspension-cultured cells and vacuolar invertase from tomato (Lycopersicon esculentum) fruit, whereas yeast invertase is not affected. However, only in the homologous system is the inhibition modulated by the concentration of Suc as previously shown for INH isolated from tobacco cells. Highly specific binding of INH to CWI could be shown by affinity chromatography of a total cell wall protein fraction on immobilized recombinant Nt-inh1 protein. RNA-blot analysis of relative transcript ratios for Nt-inh1 and CWI in different parts of adult tobacco plants revealed that the expression of both proteins is not always coordinate.

Study on the inhibitory effects of native medicinal plants on growth and aflatoxin production by toxigenic Aspergilli isolated from rainbow trout feed

In the present study, natural occurrence of fungi and aflatoxin B1 (AFB1) in pellet feed and feed ingredients used for rainbow trout was investigated with emphasis to Aspergillus section Flavi members and medicinal plants inhibitory to Aspergillus growth and/or AF production. The feed samples were cultured on the standard isolation media including dichloran rosebengal chloramphenicol agar (DRCA) and Aspergillus flavus/parasiticus agar (AFPA) for 2 weeks at 28 °C. Identification of fungal isolates was implemented based on the macro- and microscopic morphological criteria. AFs were detected using high performance liquid chromatography (HPLC). Based on the results obtained, a total of 109 fungal isolates were identified of which Aspergillus was the prominent genus (57.0%), followed by Penicillium (12.84%), Absidia (11.01%) and Pseudallscheria (10.10%). The most frequent Aspergillus species was A. flavus (60.66%) isolated from all the feed ingredients as well as pellet feed. Among 37 A. flavus isolates, 19 (51.35%) were able to produce AFB1 on yeast extract-sucrose (YES) broth in the range of 10.2 to 612.8 [tg/g fungal dry weight. HPLC analyses of trout feed showed that pellet feed and all feed ingredients tested except gluten were contaminated with different levels of AFB1 in the range of 1.83 to 67.35 lig/kg. In order to finding natural inhibitors of fungal growth and/or AF production, essential oils (EOs) and extracts of 49 medicinal plants were studied against an aflatoxin-producing A. parasiticus using a microbioassay technique. The EOs was analyzed by gas chromatography/mass spectrometry (GC/MS). Based on the results obtained, Achillea millefolium sub sp. elborsensis, Ferula gummosa, Mentha spicata, Azadirachta indica, Conium maculatum and Artemisia dracunculus remarkably inhibited A. parasiticus growth without affecting AF production by the fungus. Besides of Thymus vulgaris and Citrus aurantifolia, the EO of Foeniculum vulgare significantly inhibited both fungal growth (-70.0%) and AFs B1 and G1 (-99.0%) production. The EO of Carum carvi and ethyl acetate extract of Platycladus orientalis suppressed AFs B1 and G1 by more than 90.0%, without any obvious effect on fungal growth. The IC50 values of bioactive plants for AFs B1 and G1 were determined in the ranges of 90.6 to 576.2 and 2.8 to 61.9 µg/ml, respectively. Overall, results of the present study indicate the importance of AF contamination of trout feed as a risk factor for fish farming and thus, an urgent necessity for constant monitoring of trout feed for any unacceptable levels of AF contamination. Likewise, antifungal activities of bioactive plants introduced here would be an important contribution to explain the use of these plants as effective antimicrobial candidates to protect feeds from toxigenic fungus growth and subsequent AF contamination.

Biotechnology and crop protection in Brazil.

The process of modernization of Brazilian agriculture aimed at increasing the productivity in response to the high demand for agricultural products in the world market and it was based on the intensive use of inputs such as agrochemicals, intense mechanisation and breeding of new varieties. Among these, pesticides were incorporated in almost all production systems. Over reliance on pesticide use has produced many negative effects on both biotic and abiotic components of the environment, generating chemical contamination of soil and water, decrease in biological diversity of agroecosystems, disruption of natural cycles, pest resistance, intoxication of growers, among others. The consumption of pesticides in Brazil was 151.8 thousand tonnes in 1989, and today the country is the fifth world market of these products. The use of pesticides increased from 16 thousand tonnes (a.i.) in 1964 to 60.2 thousand tonnes in 1991, while the area planted to crops grew from 28.4 to 50.0 million ha in the same period. This means an increase of 276.2% in consumption of pesticides compared to an increase of 76% in planted area. Even with this large increase in the use of pesticides, the losses caused by pests have not been significantly reduced, and the net gain in crop productivity has been low. On the other hand, problems with food contamination, environmental degradation of growers have considerably mounted. It is possible to define two classes of crops regarding intense use of pesticides. One is represented by those crops that occupy large areas, and therefore contribute to a large amont of pesticides used for pest control in a country basis. The other class comprises crops that require large amounts of pesticides per unit of area, but not necessarily represent large amounts of pesticides used coutry-wide. Based on the classes proposed, citrus, soybean and sugarcane stand as crops with a nationally great consumption of pesticides, while tomato, potato and citrus are important as intensive users of pesticides. In this paper the biotechnologies in use, the biotechnologies in advanced stages of development, the main constraints to the development and use of biotechnlology and the impact of pesticed on the environment are discussed.

Detection and molecular characterization of Grapevine yellow speckle viroid 1 isolates infecting grapevines in Brazil.

Abstract Presently, Hop stunt viroid(HSVd) and Citrus exocortis viroid (CEVd) are the only viroids reported to infect grapevines (Vitis spp.) in Brazil, among the seven viroid species already reported infecting this host in other countries. All grapevine viroid diseases are graft-transmissible and can induce losses especiallywhenassociatedwithviruses.Theaimofthisworkwas to confirm infection by Grapevine yellow speckle viroid 1(GYSVd-1) in grapevine samples exhibiting yellow speckle symptoms in the leaves and in asymptomatic samples sequenced by next generation sequencing (NGS). The occurrence of this viroid in Brazil was further investigated in a second study. Total RNAs and dsRNAs were extracted from five symptomatic plants and 16 asymptomatic samples, respectively. Specific primers were used for RT-PCR and amplified DNA fragments were cloned and sequenced by the Sanger method. Eleven complete nucleotide sequences of GYSVd-1 isolates (366 ?367 nt) were obtained from NGS and from RT-PCR amplicons. Comparisons showed high identities (95.9 ?100 %) among ten isolates and an identity of 87.2 ?90.4 % with a divergent isolate (RM-BR). Phylogenetic analyses placed GYSVd-1 isolates in four clusters (types 1, 2, 3 and 4). All GYSVd-1 infections were confirmed by conventional RT-PCR and RT-qPCR using specific oligonucleo-tides and a labeled probe. This is the first report and molecular characterization of GYSVd-1 infecting grapevines in Brazil, and our survey indicates that this viroid could be widespread in the major grape producing regions of Brazil. Keywords GYSVd-1 . Incidence . Next generation sequencing. Secondary structure. Vine.

Essential oils and hydrolates as potential tools for integrated prevention of bacterial diseases in plants

The present work aims to investigate the potential use of natural substances against bacterial plant pathogens. Microdilution tests were therefore carried out in vitro to identify the minimum inhibitory and bactericidal concentrations (MIC and MBC) of several EOs and Hys against selected bacterial pathogens. Commercial products based on a mixture of EOs were in addition assayed with macrodilution experiments against Erwinia amylovora (Ea-causal agent of fire blight). Subsequently, using selected EOs, Hys, and commercial products, ex vivo tests on disease incidence and Ea population dynamics were carried out; the latter experiment was followed by SEM observations. In addition, in vivo resistance induction test was carried out against bacterial leaf of tomato, caused by Xanthomonas vesicatoria (Xv). EOs and Hys showed high bactericidal activity in vitro (MBC <0.1 and <10% for the most active EOs and Hys: Origanum compactum and Thymus vulgaris EOs and Citrus aurantium var. amara Hy, respectively), but they were not effective ex vivo, while resulted very active when used in vivo as resistance inducers in the tomato-Xv pathosystem (relative protection >40%). Differently, commercial products resulted active in all tests, but not as resistance inducers against Xv. An open field trial with commercial products was carried out on strawberry plants naturally infected with Xanthomonas fragariae; the results showed discrete relative protection, concerning that provided by the conventional products based on copper; mostly, the disease severity reduction on those plants treated with EOs commercial products was significant when disease severity resulted high. The papers already published described in the present work investigate (1)the activity of Hys in comparison to EOs with respect to their active volatile content; (2) the potential use of EOs and Hys in cultural heritage; for the restoration of paintings; (3) the induction of resistance caused by plasma-activated water-based root treatments.

Influence of a polyclonal antibody preparation on the in situ degradability of three energy sources

The objective of this study was to evaluate the effect of a polyclonal antibody preparation (PAP) against specific ruminal bacteria on the in situ degradability of dry-grounded maize grain (DMG), high moisture maize silage (HMMS) starch and citrus pulp (CiPu) pectin. Nine ruminally cannulated cows were used in a 3 x 3 Latin square design, replicated three times in a factorial arrangement of treatments of two rumen modifiers represented by monensin and PAP plus a control group, and the three energy sources (DMG, HMMS and CiPu). Each period had 21 days, where 16 were used for adaptation to treatment and five for data collection. The group treated with PAP showed an effect on the soluble fraction (""a"") of DMG starch, decreasing it by respectively 45.3% and 45.4% compared to the CON and MON groups. No effect of PAP was observed for any in situ degradability parameters of starch from HMMS or pectin of CiPu. It was concluded that the polyclonal antibody preparation had limited effect on the in situ degradability of the tested energy sources.

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.