Detection of mechanical and disease stresses in citrus plants by fluorescence spectroscopy

We have investigated the detection of mechanical and disease stresses in citrus plants (Citrus limonia [L.] Osbeck) using laser-induced fluorescence spectroscopy. Due to its economic importance we have chosen to investigate the citrus canker disease, which is caused by the Xanthomonas axonopodis pv. citri bacteria. Mechanical stress was also studied because it plays an important role in the plant's infection by such bacteria. A laser-induced fluorescence spectroscopy system, composed of a spectrometer and a 532 nm 10 mW excitation laser was used to perform fluorescence spectroscopy. The ratio of two chlorophyll fluorescence bands allows us to detect and discriminate between mechanical and disease stresses. This ability to discriminate may have an important application in the field to detect citrus canker infected trees. (c) 2008 Optical Society of America.

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptI1 or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease. (C) 2009 Elsevier B.V. All rights reserved.

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL-1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1-0.2 mu m2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.

CztR, a LysR-Type Transcriptional Regulator Involved in Zinc Homeostasis and Oxidative Stress Defense in Caulobacter crescentus

Caulobacter crescentus is a free-living alphaproteobacterium that has 11 predicted LysR-type transcriptional regulators (LTTRs). Previously, a C. crescentus mutant strain with a mini-Tn5lacZ transposon inserted into a gene encoding an LTTR was isolated; this mutant was sensitive to cadmium. In this work, a mutant strain with a deletion was obtained, and the role of this LTTR (called CztR here) was evaluated. The transcriptional start site of this gene was determined by primer extension analysis, and its promoter was cloned in front of a lacZ reporter gene. beta-Galactosidase activity assays, performed with the wild-type and mutant strains, indicated that this gene is 2-fold induced when cells enter stationary phase and that it is negatively autoregulated. Moreover, this regulator is essential for the expression of the divergent cztA gene at stationary phase, in minimal medium, and in response to zinc depletion. This gene encodes a hypothetical protein containing 10 predicted transmembrane segments, and its expression pattern suggests that it encodes a putative zinc transporter. The cztR strain was also shown to be sensitive to superoxide (generated by paraquat) and to hydrogen peroxide but not to tert-butyl hydroperoxide. The expression of katG and ahpC, but not that of the superoxide dismutase genes, was increased in the cztR mutant. A model is proposed to explain how CztR binding to the divergent regulatory regions could activate cztA expression and repress its own transcription.

Regulation of Catalase-Peroxidase KatG Is OxyR Dependent and Fur Independent in Caulobacter crescentus

Most organisms that grow in the presence of oxygen possess catalases and/or peroxidases, which are necessary for scavenging the H(2)O(2) produced by aerobic metabolism. In this work we investigate the pathways that regulate the Caulobacter crescentus katG gene, encoding the only enzyme with catalase-peroxidase function in this bacterium. The transcriptional start site of the katG gene was determined, showing a short 5` untranslated region. The katG regulatory region was mapped by serial deletions, and the results indicate that there is a single promoter, which is responsible for induction at stationary phase. An oxyR mutant strain was constructed; it showed decreased katG expression, and no KatG protein or catalase-peroxidase activity was detected in stationary-phase cell extracts, implying that OxyR is the main positive regulator of the C. crescentus katG gene. Purified OxyR protein bound to the katG regulatory region between nucleotides -42 and -91 from the transcription start site, as determined by a DNase I footprinting assay, and a canonical OxyR binding site was found in this region. Moreover, OxyR binding was shown to be redox dependent, given that only oxidized proteins bound adjacent to the -35 sequence of the promoter and the katG P1 promoter was activated by OxyR in an H(2)O(2)-dependent manner. On the other hand, this work showed that the iron-responsive regulator Fur does not regulate C. crescentus katG, since a fur mutant strain presented wild-type levels of katG transcription and catalase-peroxidase production and activity, and the purified Fur protein was not able to bind to the katG regulatory region.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Annual and polyetic progression of citrus canker on trees protected with copper sprays

The effects of copper sprays on annual and polyetic progress of citrus canker, caused by Xanthomonas citri subsp. citri, in the presence of the Asian citrus leafminer (Phyllocnistis citrella), were evaluated in a study conducted in a commercial orchard in northwest Parana state, Brazil, where citrus canker is endemic. Nonlinear monomolecular, logistic and Gompertz models were fitted to monthly disease incidence data (proportion of leaves with symptoms) for each treatment for three seasons. The logistic model provided the best estimate of disease progress for all years and treatments evaluated and logistic parameter estimates were used to describe polyetic disease dynamics. Although citrus canker incidence increased during each of the seasons studied, it decreased over the whole study period, more so in copper-treated trees than in water-sprayed controls. Copper treatment reduced disease incidence compared with controls in every year, especially 2004-2005, when incidence was ca. 10-fold higher in controls than in treated plots (estimated asymptote values 0 center dot 82 and 0 center dot 07, respectively). Copper treatment also reduced estimated initial disease incidence and epidemic growth rates every year.

Evaluation of two semi-selective media to detect Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds

Evaluation of two semi-selective media to detect Curtobacterium flaccumfaciens pv. flaccumfaciens in bean seeds This study aimed to compare the effectiveness of the semi-selective MSCFF and modified CNS culture media in detecting Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) in bean seeds, using the streak and spread plate techniques. Four 500 g subsamples, obtained from two samples of bean seeds, were immersed in 600 mL of sterile distilled water for 18 h at 5 degrees C. Suspensions were picked and transferred to plates with both culture media. Plates were then incubated at 28 degrees C, and bacterial growth on both media was evaluated 72 and 144 hours later, compared to the growth of a Cff reference strain. Both media revealed the presence of Cff colonies. Typical colonies were isolated for PCR analyses and pathogenicity tests on tobacco leaves. A characteristic Cff growth on MSCFF medium was observed for the seed samples, for the two plate techniques used, in both evaluations. On the modified CNS culture medium, the bacterial growth was only detected in seed samples after 144 hours of incubation, regardless of the plate technique used. The results showed Cff grew faster on the MSCFF semi-selective culture medium. Bacterial isolates tested were identified as Cff by both PCR analyses and a positive tobacco hypersensitivity reaction.

Replacement of the arginine biosynthesis operon in Xanthomonadales by ateral gene transfer

The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the gamma-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.

Efeito residual da silicatagem no solo e na produtividade do capim-marandu sob pastejo

A escória siderúrgica é uma alternativa para a correção da acidez dos solos e é constituída de silicato de cálcio. Neste estudo, avaliaram-se os efeitos residuais da aplicação de silicato de cálcio nos atributos químicos do solo e da planta em Latossolo Vermelho distroférrico típico com capim-Marandu (Brachiaria brizantha cv. Marandu), sob intensidades de pastejo em lotação rotacionada. O delineamento experimental foi de blocos ao acaso, com intensidades de pastejo avaliadas pelas ofertas diárias de forragem de 50, 100, 150 e 200 kg t-1 de MS por PV nas parcelas experimentais, enquanto a aplicação superficial de silicato de cálcio combinado com calcário dolomítico, respectivamente, nas doses 0 + 0; 2 + 0; 4 + 0; 6 + 0; 2 + 4; 4 + 2 e 0 + 6 t ha- 1 nas subparcelas com quatro repetições, duas épocas (verão e inverno) e avaliação em três profundidades do solo (0-10, 10-20 e 20-40 cm). Os atributos químicos do solo pH em CaCl2, Ca, Mg, K, H + Al e V, avaliados 720 dias após a aplicação, apresentaram resultados favoráveis do poder residual do silicato de Ca e do calcário. A oferta de forragem 200 kg t-1 e o tratamento somente com calcário dolomítico (0 + 6 t ha-1) elevaram o valor de pH em CaCl2 e o V, principalmente na camada de 0-10 cm. Os teores de Si no solo foram influenciados pelas doses aplicadas de silicato de Ca, apesar de não terem causado alterações significativas nos teores foliares de Si. A composição químico-bromatológica foi afetada somente pelas ofertas e épocas. As ofertas, épocas e a interação época x oferta resultaram em efeitos na produção de matéria seca no pré-pastejo, com maiores produções para a oferta 200 kg t-1 e menores para a de 50 kg t-1 nas duas épocas. O resíduo (pós-pastejo) foi influenciado pelas ofertas e épocas. As ofertas 50 e 100 kg t-1 e o tratamento com 2 t ha-1 de silicato de Ca promoveram as maiores taxas de acúmulo de matéria seca.

Avaliação dos fluxos das valvas aórtica e pulmonar com ecocardiografia Doppler pulsátil em cães clinicamente sadios

Avaliaram-se com Doppler pulsátil os fluxos das valvas aórtica (AO) e pulmonar (Pul) por meio de análise qualitativa (presença de regurgitações valvares e características do espectro avaliado) e quantitativa, com obtenção de parâmetros ecocardiográficos como velocidades máxima (V. Max.) e média (V. Me.), integral de velocidade (VTI), tempo de aceleração (TA) e ejeção (TE), de 30 cães considerados clinicamente sadios por meio de exames físico, laboratoriais, eletrocardiográfico, ecocardiográfico (modos uni e bidimensional), radiográfico de tórax e mensuração da pressão arterial sistêmica. Obtiveram-se os seguintes resultados para os referidos parâmetros: V.max. AO= 1,22± 19,38m/s; V. Me. AO= 0,72± 0,08m/s; VTI AO= 0,14± 0,02m; TA AO= 38,80± 11,29ms; TE AO= 197,90± 24,77ms; V. Max. Pul= 0,95± 0,18m/s; V. Me. Pul= 0,63± 0,10m/s; VTI Pul= 0,13± 0,02m; TA Pul= 70,97± 18,87ms; TE Pul= 203,70± 28,98ms. Em apenas três animais observou-se regurgitação pulmonar. Alguns parâmetros apresentaram correlação negativa com a variável freqüência cardíaca (VTI AO, TE AO, VTI Pul, TA Pul, TE Pul); outros correlação positiva com a variável peso (VTI AO, TA AO, TE AO, VTI Pul, TE Pul,) e não se observou influência da variável sexo na maioria dos parâmetros avaliados. Na comparação entre os dois fluxos, observaram-se V. Max. AO e V. Me. AO maiores que V. Max. Pul. e V. Me. Pul., respectivamente, VTI AO maior que VTI Pul, e TA AO menor que TA Pul.

Acurácia, precisão e robustez das estimativas da digestibilidade aparente da matéria seca determinada com o uso de indicadores em ovinos

O objetivo foi avaliar a acurácia, precisão e robustez das estimativas da digestibilidade aparente da matéria seca obtidas utilizando-se como indicadores fibra em detergente ácido indigestível (FDAi), fibra em detergente neutro (FDNi) indigestível, lignina em detergente ácido (LDA), LDA indigestível (LDAi) e óxido crômico em comparação ao método de coleta total de fezes. Dezoito ovinos (56,5 ± 4,6 kg PV) foram designados aleatoriamente a dietas compostas de 25, 50 ou 75% de concentrado e feno de Coast cross por 25 dias. As fezes foram coletadas por cinco dias para determinação da digestibilidade aparente da MS. As amostras de alimentos e fezes foram incubadas no rúmen de três bovinos por 144 horas, para obtenção das frações indigestíveis. Óxido crômico foi administrado a 4,0 g/animal/dia. A acurácia foi avaliada pela comparação do viés médio (DAMS predito - DAMS observado) entre os indicadores; a precisão, por meio da raiz quadrada do erro de predição e do erro residual; e a robustez, pelo estudo da regressão entre o viés e o consumo de matéria seca, o nível de concentrado e o peso vivo. A recuperação fecal e a acurácia das estimativas da digestibilidade aparente da MS foram maiores para FDAi, seguida pela FDNi, LDAi, pelo óxido crômico e depois pela lignina em detergente ácido. O viés linear foi significativo apenas para FDAi, FDNi e LDAi. O uso de óxido crômico permitiu estimativas mais precisas da digestibilidade aparente da MS. Todos os indicadores foram robustos quanto à variação no consumo de matéria seca e apenas LDAi e óxido crômico foram robustos quanto aos níveis de concentrado na dieta. O óxido crômico não foi robusto quando houve variação no peso vivo animal. Assim, a FDAi é o indicador mais recomendado na estimativa da digestibilidade aparente da MS em ovinos quando o objetivo é comparar aos dados da literatura, enquanto o óxido crômico é mais recomendado quando o objetivo é comparar tratamentos dentro de um mesmo experimento.

Efeitos da enramicina e monensina sódica sobre a digestão de nutrientes em bovinos alimentados com dietas contendo alto nível de concentrados

Estudaram-se os efeitos da administração de enramicina e monensina sódica sobre a digestão total dos nutrientes da dieta e o consumo de matéria seca digestível em bovinos. Doze fêmeas bovinas não-gestantes e não-lactantes (675 ± 63 kg PV) foram distribuídas inteiramente ao acaso em três tratamentos (controle, enramicina e monensina) e alimentados com dieta contendo 60% de concentrados (milho, farelo de soja e minerais) e 40% de volumoso (cana-de-açúcar). A enramicina foi administrada na dose de 20 mg/animal/dia e a monensina na dose de 300 mg/animal/dia. O experimento teve duração total de 21 dias, de modo que os últimos dez dias foram utilizados para administração do marcador externo (15 g de óxido crômico/animal/dia) e os últimos cinco dias para a coleta de fezes e amostragem dos alimentos. Nenhum dos antibióticos alterou os consumos de matéria seca digestível e NDT e a digestibilidade de matéria seca, proteína bruta, extrato etéreo, fibra em detergente ácido, fibra em detergente neutro, amido, energia bruta e nutrientes digestíveis totais.

Plasmodium vivax: Induction of CD4(+)CD25(+)FoxP3(+) Regulatory T Cells during Infection Are Directly Associated with Level of Circulating Parasites

Circulation CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been associated with the delicate balancing between control of overwhelming acute malaria infection and prevention of immune pathology due to disproportionate inflammatory responses to erythrocytic stage of the parasite. While the role of Tregs has been well-documented in murine models and P. falciparum infection, the phenotype and function of Tregs in P. vivax infection is still poorly characterized. In the current study, we demonstrated that patients with acute P. vivax infection presented a significant augmentation of circulating Tregs producing anti-inflammatory (IL-10 and TGF-beta) as well as pro-inflammatory (IFN-gamma, IL-17) cytokines, which was further positively correlated with parasite burden. Surface expression of GITR molecule and intracellular expression of CTLA-4 were significantly upregulated in Tregs from infected donors, presenting also a positive association between either absolute numbers of CD4(+)CD25(+)FoxP3(+)GITR(+) or CD4(+)CD25(+)FoxP3(+)CTLA-4(+) and parasite load. Finally, we demonstrate a suppressive effect of Treg cells in specific T cell proliferative responses of P. vivax infected subjects after antigen stimulation with Pv-AMA-1. Our findings indicate that malaria vivax infection lead to an increased number of activated Treg cells that are highly associated with parasite load, which probably exert an important contribution to the modulation of immune responses during P. vivax infection.