Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach / Introdução do gene resistente à ferragem da folha bacteriana (BLB), xa7 na variedade MARDI elite, MR219 por abordagem de backcrossing assistida por marcador (MABC)

Abstract Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Resumo A mancha bacteriana das folhas (BLB) é uma das principais doenças do arroz na Malásia. Essa doença causa perdas substanciais de rendimento de até 70%. O desenvolvimento de variedades de arroz que herdaram características de resistência ao BLB é uma abordagem crucial para promover e sustentar a indústria do arroz na Malásia. Portanto, o objetivo deste estudo foi aumentar os caracteres BLB resistentes a doenças da variedade comercial MR219 de alto rendimento por meio de uma abordagem de cruzamento retrocruzamento com ferramenta de apoio de seleção assistida por marcador (MAS). O gene de resistência a BLB de amplo espectro, Xa7 do pai doador IRBB7, foi introgressado no MR219 suscetível (pai recorrente) usando dois marcadores flanqueadores ID7 e ID15. No BC3F4, conseguimos gerar 19 linhagens introgressadas com o gene Xa7 homozigoto e apresentamos características de resistência como genitor doador quando desafiado com Xanthomonas oryzae pv. oryzae por inoculação artificial. O pai recorrente MR219 e a variedade controle, MR263, estavam gravemente infectados pela doença. As linhas melhoradas exibiram características morfológicas e de desempenho de rendimento semelhantes às da variedade elite, MR219. Duas linhas, PB-2-107 e PB-2-34, foram escolhidas como linhas potenciais por causa de seus desempenhos excelentes em comparação com a mãe, MR219. Este estudo demonstra uma história de sucesso de aplicação de MAS no desenvolvimento de linhas de arroz melhoradas com resistência a doenças contra a doença BLB.

Antagonistic activity and application of Bacillus velezensis strain Bv-303 against rice bacterial-blight disease caused by Xanthomonas oryzae pv. oryzae / 生物工程学报

In this study, a new Bacillus velezensis strain Bv-303 was identified and its biocontrol effect against rice bacterial-blight (BB) disease caused by Xanthomonas oryzae pv. oryzae (Xoo) was investigated. Cell-free supernatant (CFS) of strain Bv-303 under different growth conditions were prepared to test the antagonistic activity and stability against Xoo by the Oxford-cup method in vitro. The antibacterial effect of strain Bv-303 to BB disease in rice were further analyzed in vivo by spraying the cell-culture broth (CCB), CFS and cell-suspension water (CSW), respectively, on the rice leaves inoculated with Xoo. Additionally, rice seeds germination rate and seedling growth under the strain Bv-303 CCB treatment were tested. The results showed that the strain Bv-303 CFS significantly inhibited Xoo growth by 85.7%‒88.0% in vitro, which was also stable under extreme environment conditions such as heat, acid, alkali and ultraviolet light. As tested in vivo, spraying the CCB, CFS or CSW of strain Bv-303 on the Xoo-infected leaves enhanced rice plant resistance to BB disease, with CCB showing the highest increase (62.7%) in disease-resistance. Notably, CCB does not have negative effects on rice seed germination and seedling growth. Therefore, strain Bv-303 has great potential for biocontrol of the rice BB disease.

Resistance to diseases of sour, sweet and wild passion fruit genotypes

The cultivation of passion fruit is important for Brazil, since the country is currently the largest producer and consumer of fruit in the world. However, the fields of passion fruit still face important problems due to the incidence and severity of diseases in the field. Thus, the present study aimed to assess resistance to bacterial and fungal diseases in 13 genotypes of sour, sweet and wild passion fruit, in field conditions in the Distrito Federal, Brazil. For this, a field experiment was installed in a randomized block design, with four replications and 13 treatments (genotypes). The characteristics of incidence, severity and degree of resistance for bacteriosis, septoriosis, scab and anthracnose diseases were evaluated in 5 fruits per plot of each genotype. Genetic parameters of the evaluated traits were also estimated. High heritability values and CVg/Cve ratio were observed for most of the evaluated characteristics. The genotypes presented mean values of incidence and severity of bacteriosis, septoriosis, scab and anthracnose different among them, and the one that presented the best results in the degree of resistance for all diseases was F1 (MAR20 # 24 x ECL7 P1 R4).

Screening and formulation of novel carriers for Xanthomonas bacteriophage to control bacterial leaf blight disease

Aims@#This study was aimed to evaluate the potential of several carriers to formulate the phages and retain their activity under various pH and temperature conditions.@*Methodology and results@#The skim milk, rice flour, corn flour and CalnuXan (calcium and magnesium) as carriers to formulate the isolated phage to maintain its activity under extreme pH and temperature conditions. Two phages formulated with carriers retained their viability at pH 5, pH 7 and pH 9 compared to that of the unformulated phages. Besides, the formulated phages also retained a high titre compared to the unformulated phages when they were exposed to 37 °C and 45 °C. Based on the in vitro study of the formulation, it was applied in the glass house. The plant height, leaf chlorophyll and disease scoring were recorded and analyzed. In the glass house, the rice plant treated with formulated phages showed higher plant height and chlorophyll content than those treated with unformulated or untreated phages. Nonetheless, both formulated and unformulated protected the rice plant, which showed lower disease severity than the untreated group.@*Conclusion, significance and impact of study@#Phage therapy has been used for treating plant diseases caused by pathogenic bacteria. Despite their effectiveness in killing the pathogen in vitro, the results were not reproducible in the field. Bacteriophages (phages) are sensitive to environmental factors and infection efficiency was dropped when exposed to harmful environments. However, this study successfully formulated two novels Xanthomonas phages, as biocontrol agents against bacterial leaf blight (BLB) disease in rice.

Suppression of Xanthomonas oryzae pv. oryzae biofilm formation by Acacia mangium methanol leaf extract / Supressão de Xanthomonas oryzae pv. oryzae: formação do biofilme oryzae pelo extrato de folhas de metanol de Acacia mangium

Abstract Xanthomonas oryzae pv. oryzae (Xoo), a pathogen responsible for rice bacterial leaf blight, produces biofilm to protect viable Xoo cells from antimicrobial agents. A study was conducted to determine the potency of Acacia mangium methanol (AMMH) leaf extract as a Xoo biofilm inhibitor. Four concentrations (3.13, 6.25, 9.38, and 12.5 mg/mL) of AMMH leaf extract were tested for their ability to inhibit Xoo biofilm formation on a 96-well microtiter plate. The results showed that the negative controls had the highest O.D. values from other treatments, indicating the intense formation of biofilm. This was followed by the positive control (Streptomycin sulfate, 0.2 mg/mL) and AMMH leaf extract at concentration 3.13 mg/mL, which showed no significant differences in their O.D. values (1.96 and 1.57, respectively). All other treatments at concentrations of 6.25, 9.38, and 12.5 mg/mL showed no significant differences in their O.D. values (0.91, 0.79, and 0.53, respectively). For inhibition percentages, treatment with concentration 12.5 mg/mL gave the highest result (81.25%) followed by treatment at concentrations 6.25 and 9.38 mg/mL that showed no significant differences in their inhibition percentage (67.75% and 72.23%, respectively). Concentration 3.13 mg/mL resulted in 44.49% of biofilm inhibition and the positive control resulted in 30.75% of biofilm inhibition. Confocal laser scanning microscopy (CLSM) analysis of Xoo biofilm inhibition and breakdown showed the presence of non-viable Xoo cells and changes in aggregation size due to increase in AMMH leaf extract concentration. Control slides showed the absence of Xoo dead cells.

Resumo Xanthomonas oryzae pv. oryzae (Xoo), um patogênico responsável pela influência bacteriana na folha do arroz, produz biofilme para proteger células Xoo viáveis de agentes antimicrobianos. Foi conduzido um estudo para determinar a potência do extrato de folha de Acacia mangium methanol (AMMH) como um inibidor de biofilme Xoo. Quatro concentrações (3,13, 6,25, 9,38 e 12,5 mg/mL) de extrato de folha de AMMH foram testadas quanto à sua capacidade de inibir a formação de biofilme Xoo em uma placa de microtitulação de 96 poços. Os resultados mostraram que os controles negativos tiveram o maior valor de OD do que os outros tratamentos, indicando a intensa formação de biofilme. Isso foi seguido do controle positivo (sulfato de estreptomicina, com concentração de 0,2 mg/mL, e extrato de folha de AMMH, com concentração de 3,13 mg/mL), que não apresentou diferenças significativas nos seus valores OD (1,96 e 1,57, respectivamente). Todos os outros tratamentos com concentrações de 6,25, 9,38, e 12,5 mg/mL não tiveram diferenças significativas nos seus valores OD (0,91, 0,79, e 0,53, respectivamente). Para percentagens de inibição, o tratamento com concentração 12,5 mg/mL apresentou o maior resultado (81,25%), seguido do tratamento em concentrações de 6,25 e 9,38 mg/mL, que não mostraram diferenças significativas na sua percentagem de inibição (67,75 e 72,23%, respectivamente). Concentração 3,13 mg/mL resultou em 44,49% de inibição do biofilme, e o controle positivo resultou em 30,75% de inibição do biofilme. Análise por microscopia confocal de leitura a laser de inibição e separação de biofilme Xoo revelou a presença de células Xoo não viáveis e alterações no tamanho da agregação por causa do aumento na concentração de extrato de folha de AMMH. Slides de controle mostraram a ausência de células Xoo mortas.

Application of Films Based on Chitosan and Xanthan Gum in Refrigerated Fish Conservation

Abstract This research aims to determine the efficiency of chitosan and xanthan gum films in conservation of croaker fillets kept in refrigeration for 9 days. Proximal composition, loss of mass, color, pH, TVB-N (Total Volatile Bases) and microbiological profile were assessed. The films were prepared with chitosan and xanthan gum in varying mass proportions 100:0, m:m (C100XG0); 60:40, m:m (C60XG40); 50:50, m:m (C50XG50). They presented the respective values for moisture content, water solubility, thickness and water vapor permeability: 24.59%, 19.50%, 0.086 mm and 11.45gm-1.s-1.Pa-1for C100XG0; 24.58%; 20.27%, 0.091 mm and 10.41 gm-1.s-1.Pa-1for C60XG40; 22.11%, 22.06%, 0.089 mm and 10.68 gm-1.s-1.Pa-1 forC50XG50.The films were made in small bags format capable to hold about 20 g of fish fillets. A control sample was prepared in parallel, using polyethylene bags under the same storage conditions. The results showed that the chitosan films combined with xanthan gum had excellent antimicrobial properties, capable of preserving the quality of chilled fish fillets during the studied period, since it inhibited the growth of Staphylococcus coagulase-positive, Salmonella spp and coliforms at 45 ° C. Mass loss of the croaker fillets was not significantly affected by xanthan gum addition to the films. On the other hand, xanthan gum addition affected pH and color parameters of the corvina fillets. It was also verified that the combination of these two polymers promoted the reduction of N-BVT, being the C50XG50 film that presented the best response.

Development and validation of a standard area diagram set as assessment aid for estimating the severity of bacterial spot on tri-lobed leaves of yellow passion fruit / Desenvolvimento e validação de escala diagramática como auxílio na avaliação da severidade da bacteriose em folhas trilobadas do maracujazeiro azedo

Bacterial spot (Xanthomonas axonopodis pv. passiflorae) significantly reduces yellow passion fruit (Passiflora edulis Sims) yield and longevity. A standard area diagram set (SADs) for severity assessment of bacterial spot on tri-lobed leaves of yellow passion was developed and validated in this study. The SADs consisted of eight severity levels (2; 4; 9; 18; 35; 58; 80; and 94%). For its validation, 20 raters, who initially estimated the disease severity without the aid of the SADs, were divided into four groups (G1 and G3, inexperienced; G2 and G4, experienced). Subsequently, G1 and G2 performed the second evaluation without the SADs, and G3 and G4 completed the second evaluation with the proposed SADs. The accuracy and precision of the assessments were determined by simple linear regression and by the Lin's concordance correlation coefficient (LCCC). The proposed SADs allowed accurate and precise quantification of bacterial spot severity, increasing the agreement between estimated and actual values. Inexperienced raters benefited the most from the use of the SADs. The increase in accuracy and precision in the non-aided groups, when present, was less pronounced than those increments observed in the SADs-aided groups. The LCCC confirmed the increases in accuracy and precision detected by the linear regression analysis.

A bacteriose (Xanthomonas axonopodis pv. passiflorae) reduz significativamente a produção e longevidade do maracujazeiro azedo (Passiflora edulis Sims). Uma escala diagramática para a avaliação da severidade da bacteriose em folhas trilobadas do maracujazeiro azedo foi desenvolvida e validada neste estudo. A escala diagramática apresentou oito níveis de severidade (2; 4; 9; 18; 35; 58; 80 e 94%). Para a sua validação, os 20 avaliadores foram divididos em quatro grupos (G1 e G3, sem experiência; G2 e G4, com experiência), que inicialmente estimaram a severidade da doença sem auxílio da escala. Posteriormente, G1 e G2 fizeram outra avaliação sem escala, e G3 e G4 realizaram a avaliação com a escala proposta. A acurácia e a precisão das estimativas foram determinadas por regressão linear simples e pelo coeficiente de correlação de concordância de Lin (LCCC). A escala diagramática proposta permitiu quantificar a severidade da bacteriose de forma acurada e precisa, aumentando a concordância entre os valores estimados e os reais. Os avaliadores inexperientes foram os mais beneficiados pelo uso da escala. O aumento da acurácia e precisão nos grupos que realizaram dupla avaliação sem escala, quando ocorreu, foi mais discreto que os incrementos observados nos grupos que utilizaram a escala. O LCCC confirmou os incrementos da acurácia e precisão detectados pela análise de regressão linear.

Gene expression analysis identifies hypothetical genes that may be critical during the infection process of Xanthomonas citri subsp. citri

Background: Gene expression analysis via microarray is widely used in phytobacteria to validate differential gene expression associated with virulence or to compare biological profiles of wild type and mutant strains. Here, we employed DNA microarrays to study the early stages of the infection process (24, 72 and 120 h post-inoculation) of Xanthomonas citri subsp. citri (Xac) infecting Citrus sinensis to interrogate the expression profiles of hypothetical genes. Results: Under infective conditions, 446 genes were up- and 306 downregulated. Outstanding among genes upregulated during infection were those involved in synthesizing the Type 3 Secretion System and effectors, xanthan gum and quorum-sensing induction, and flagellum synthesis and regulation. Additionally, 161 hypothetical genes were up- and 100 were downregulated, 49 of which are known to have a significant biological role. To understand hypothetical gene co-regulation or -expression, nine expression profiles including 158 genes were identified during the three infection phases. Of these, 47 hypothetical genes were identified as having expression profiles associated with at least one connected to a gene associated with adaptation and virulence. Conclusions: Expression patterns of six differentially expressed genes were validated by quantitative reverse transcription polymerase chain reaction, thus demonstrating the effectiveness of this tool in global gene expression analysis in Xac.

Xanthan gum production by Xanthomonas axonopodis pv. mangiferaeindicae from glycerin of biodiesel in different media and addition of glucose

Biodiesel production has been increasing yearly in Brazil. A large amount of glycerin is generated in this process and needs a correct destination. One possible use of this glycerin in crude form is in biotechnological processes. Xanthan gum is a commercial gum used primarily in the pharmaceutical and food industries as thickener, emulsifier and stabilizer. It is synthetized by species of the bacterium Xanthomonas generally from glucose. However, current research shows that species of this bacterium have the capacity to grow and synthesize the gum using glycerin from biodiesel. The aim of this study was to produce xanthan gum using glycerin from biodiesel production in medium with different nitrogen content, named complex and simple media. The kinetics of fermentation in simple medium showed a twofold increase in gum production (3.16 kg.m-3) compared to the one in complex medium (1.46 kg.m-3) after 120 hours. The gum generated in this study showed chemical and rheological characteristics of xanthan gum. Glucose supplementation did not show an increase in xanthan production but did increase the consistency index and the behavioral index of solutions of this gum.

Caracterização funcional de sistemas de dois componentes em Xanthomonas citri / Functional characterization of two-component systems in Xanthomonas citri

Xanthomonas citri subsp. citri, é uma bactéria pertencente à classe das Gamaproteobactérias, fitopatogênica, que exibe uma especificidade patógeno-hospedeiro extremamente alta. X. citri infecta plantas do gênero Citrus, causando o cancro cítrico, uma doença destrutiva encontrada em cultivos ao redor do mundo. Esta bactéria apresenta em seu genoma 34 genes que codificam proteínas relacionadas com o metabolismo do segundo mensageiro c-di-GMP. Em geral, níveis elevados de c-di-GMP favorecem a sessilidade e a produção de exopolissacarídeos, enquanto níveis mais baixos resultam em maior motilidade e aumento na dispersão do biofilme. Com o intuito inicial de buscar novos alvos de X. citri que dependessem dos níveis intracelulares desse segundo mensageiro, foram analisados os proteomas de linhagens mutantes em diguanilato ciclases específicas. Nas análises proteômicas por eletroforese bidimensional foram identificadas 15 proteínas diferencialmente expressas presentes em mais de um dos proteomas dos mutantes analisados. Entre estas, duas proteínas reguladoras de resposta e preditas de participar de sistemas de dois componentes, XAC0834 e XAC3443, foram encontradas sendo mais expressas em mutantes que apresentavam fenótipo de alto c-di-GMP; enquanto uma proteína hipotética provavelmente presente na membrana, XAC3657, estava mais expressa em linhagens com fenótipos relacionados a baixos níveis de c-di-GMP. Por meio de uma análise por qRT-PCR foi verificado que os níveis de mRNA para XAC0834 e XAC3443 não variam entre as linhagens e, portanto, a diferença nos níveis de expressão destas proteínas deve ocorrer póstranscricionalmente. Como os sistemas de dois componentes e proteínas de membrana são importantes para a adaptação das bactérias a diferentes condições ambientais, o objetivo do presente trabalho foi a caracterização funcional de XAC0834, XAC3433 e XAC3657, com maiorênfase em XAC0834 e na provável proteína sensora cognata, XAC0835, de forma a contribuir para a melhor compreensão dos processos de regulação da virulência de bactérias. Na análise da organização gênica dos genes que codificam estas proteínas, foi verificado que os genes XAC0834 e XAC0835 formam um operon, juntamente com a tioesterase XAC0833 e, portanto, o nível transcricional destes genes ocorre pelos mesmos reguladores, apoiando a hipótese de se tratarem de um sistema de dois componentes; assim como os genes XAC3442 e XAC3443. Utilizando uma linhagem mutante em XAC0834, mostramos que esta proteína impacta positivamente a motilidade sliding e a formação de biofilme, e tem efeito contrário no crescimento de X. citri em meio rico 2xTY e na motilidade twitching. Como estes fenótipos são modulados por c-di-GMP, é possível que a deleção deste gene altere significativamente os níveis de c-di-GMP nas células. Além disto, foi verificado que as proteínas XAC0835, XAC3443 e XAC3657 não afetam a motilidade sliding, mas, individualmente, XAC0835 é importante para a formação de biofilme; XAC3657 afeta negativamente o crescimento de X. citri em meio rico 2xTY; e XAC3443 afeta negativamente a motilidade twitching. Na análise do transcritoma da superexpressão de XAC0834, foi observado que havia aumento na expressão de genes relacionados ao sistema de secreção do tipo IV e na montagem do pilus do tipo IV, em comparação com a linhagem selvagem, o que pode estar relacionado aos fenótipos observados. Este trabalho forneceu subsídios importantes para a compreensão do papel fisiológico do sistema de dois componentes XAC0834/XAC0835, assim como do regulador de resposta XAC3443 e da proteína hipotética, XAC3657, em X. citri, o que pode contribuir para o entendimento da relação de c-di-GMP com os sistemas de dois componentes

Xanthomonas citri subsp. citri, is a phytopathogenic Gammaproteobacteria, with extremely high pathogen-host specificity. X. citri infects plants of the genus Citrus, causing citrus canker, a destructive disease found in crops around the world. The genome of X. citri pv. citri 306 (XAC 306) contains 34 genes encoding proteins related to the second messenger c-di-GMP metabolism. In general, high levels of c-di-GMP favor the sessility and exopolysaccharide production, whereas lower levels result in greater motility and increased biofilm dispersion. In order to initially search for new X. citri targets that depend on the intracellular levels of this second messenger, the proteomes of specific diguanylate cyclase mutant strains were analyzed by two-dimensional electrophoresis. Fifteen differentially expressed proteins present in more than one of the mutant proteomes compared to wild type were identified. Among these, two proteins predicted to participate as response regulators in two-component systems, XAC0834 and XAC3443, were found to be more expressed in mutants with high c-di-GMP phenotypes; whereas a hypothetical membrane protein, XAC3657, was more expressed in strains with low cdi-GMP-related phenotypes. Relative mRNA levels for XAC0834 and XAC3443, as determined by qRT-PCR, do not vary among the analyzed strains, suggesting post-transcriptional regulation. Because two-component systems and membrane proteins are important for the adaptation of bacteria to different environmental conditions, the aim of this work was the functional characterization of XAC0834, XAC3433 and XAC3657, with greater emphasis on XAC0834 and its probable cognate sensor protein, XAC0835, contributing to a better understanding of the processes of bacterial virulence regulation. Genes XAC0834 and XAC0835 form an operon, together with the XAC0833 coding for a thioesterase, suggesting that they are co-regulated, aswell as the XAC3442 and XAC3443 genes. Using a mutant strain in XAC0834, we show that this protein positively impacts sliding motility and biofilm formation and has the opposite effect on X. citri growth in rich medium and twitching motility. Because these phenotypes are modulated by c-di-GMP, deletion of this gene may alter cellular c-di-GMP levels. In addition, we found that XAC0835, XAC3443 and XAC3657 proteins do not affect sliding motility, but XAC0835 is important for biofilm formation; XAC3657 negatively affects X. citri growth in rich medium; and XAC3443 negatively affects twitching motility. The RNA-seq transcriptome of X. citri overexpressing XAC0834 was compared to the control strain, and there was an increase in the expression of genes for the type IV secretion system and the assembly of the type IV pilus, which may be related to the observed phenotypes. This work provided important insights for understanding the physiological role of the XAC0834/XAC0835 two-component system as well as the XAC3443 response regulator and the hypothetical protein XAC3657, in X. citri which may contribute to the understanding of the relationship of c- di-GMP with two-component systems

Molecular methods for diversity assessment among xanthomonads of Bulgarian and Macedonian pepper

ABSTRACT Bacterial spot is an important disease of pepper in Bulgaria and Macedonia. For characterization of Xanthomonas species associated with bacterial spot, 161 strains were collected from various field pepper-growing regions. Among them, 131 strains were identified as Xanthomonas euvesicatoria and 30 as Xanthomonas vesicatoria using species-specific primers and polymerase chain reaction followed by restriction fragment length polymorphism analysis. To assess the genetic diversity of the strains, two methods (Random Amplified Polymorphic DNA and Repetitive Element Palindromic-Polymerase Chain Reaction) were applied. Discriminatory index was calculated and analysis of molecular variance was carried out.Combined random amplified polymorphic DNA analysis of the X. euvesicatoria strains with primers CUGEA-4 and CUGEA-6 had greater discriminative power (0.60) than repetitive element palindromic-polymerase chain reaction with ERIC and BOX A1R primers, which makes this method applicable for strain diversity evaluation. Discrimination among the X. vesicatoria strains was achieved by the use of ERIC primers and only for the Bulgarian strains. The results demonstrated that X. euvesicatoria was more diverse than X. vesicatoria and heterogeneity was observed mainly in the Bulgarian populations. According to the analysis of molecular variance, genetic variations in X. euvesicatoria were observed among and within populations from different regions, while the differences between the two countries were minor. Following the principal coordinates analysis, a relation between the climatic conditions of the regions and a genetic distance of the populations may be suggested.

Caracterização de dois pares efetor/inibidor associados ao sistema de secreção tipo IV de Xanthomonas citri / Characterization of the two effector/inhibitor pair associated with the type IV secretion system of Xanthomonas citri

O sistema de secreção tipo IV (T4SS) da família de bactérias Xanthomonadaceae transfere efetores (X-Tfes) com a capacidade de matar outras bactérias, conferindo uma vantagem em comunidades bacterianas mistas para colonizar diferentes nichos como o solo ou as superfícies das plantas. Os X-Tfes possuem diferentes domínios putativos com atividades hidrolíticas contra componentes do envelope celular bacteriano do tipo: glicohidrolases, transglicosilases, amidases e lipases. Os X-Tfes por sua atividade biológica inata podem ocasionar dano intracelular para a bactéria que os produz. Para se proteger contra estas atividades, também são produzidas lipoproteínas com função inibitoria (X-Tfis) localizadas no periplasma. Os genes que codificam os X-Tfes e os X-Tfis estão organizados em operons, o que permite gerar os pares efetor/inibidor simultaneamente. Entre os potenciais X-Tfes do fitopatógeno Xanthomonas citri estão Xac1918 e Xac0574. Xac1918 é uma proteína com um domínio da superfamília da lisozima e um domínio conhecido como RTX (Repeats in Toxin) de ligação ao cálcio, enquanto Xac0574 tem um domínio da superfamília da lipase 3. Os seus possíveis inibidores, Xac1917 e Xac0573 respectivamente, apresentam um peptídeo sinal no N-terminal contendo o lipobox representativo das lipoproteínas. As proteínas Xac0574 e Xac0573 são monômeros em solução que formam um complexo estável 1:1, favorecido termodinamicamente (ΔG°= -12 Kcal/mol) com uma constante de dissociação de 2,45 nM, garantindo que a bactéria fique protegida contra os efeitos nocivos de Xac0574 quando é produzida intracelularmente. Xac0574 é uma fosfolipase A1, sem atividade lisofosfolipase, com a capacidade de hidrolisar os três fosfolipídios majoritários que compõem a membrana celular bacteriana, fosfatidilglicerol (PG), cardiolipina e fosfatidiletanolamina (PE), mostrando uma aparente preferência pelo último. A atividade enzimática de Xac0574 explica a forte inibição do crescimento celular em E. coli após da sua indução heteróloga, já que gera uma diminuição de quase 10 vezes da população celular comparada com a cultura não induzida com a mesma construção. Poroutro lado, Xac0573 inibe efetivamente a atividade enzimática de Xac0574 ao formar o complexo, além de não ter atividade fosfolipase nem lisofosfolipase. Foram produzidos cristais da Xac1918 e Xac0573 que difrataram com uma resolução de 3,0 e 2,5 Å, respectivamente. Porém, só foi gerado um modelo de Xac0573. Xac0573 está composta por duas folhas ß antiparalelas com uma topologia característica de ß sanduíche Com uma pequena hélice e duas voltas. Um alinhamento de homólogos de Xac0573 identificou nas extremidades da proteína as regiões conservadas, constituindo duas possíveis interfaces de interação que podem ser as responsáveis por bloquear o acesso dos fosfolipídios ao sítio catalítico ou impedir os rearranjos estruturais de Xac0574 que são necessários para a sua atividade enzimática. Adicionalmente, a topologia da Xac0573 é semelhante do domínio C2, conhecido em eucariotos como domínio de ligação ao lipídio e ao cálcio, e está envolvido em processos de sinalização de segundos mensageiros lipídicos, proteínas de trafego de membranas e mecanismos de fusão de membranas. Nossos resultados apontam para uma nova função biológica do domínio C2 como um inibidor enzimático intracelular em bactérias

The type IV secretion system (T4SS) of the bacteria family Xanthomonadaceae transfers effectors (X-Tfes) with that can kill other bacterial cells, conferring an advantage to the bacterial community during colonization of different niches in the soil or on the plant surface. The X-Tfes possess different putative domains with hydrolytic activity against components of the bacterial cellular envelope, including glycohydrolase, transglycolase, amidase and lipase domain. The innate biological activity of X-Tfes can cause intracellular damage. Therefore, the bacteria that produce them also produce lipoproteins with inhibitor function (X-Tfis) located in the periplasm for their protection. The genes that code for X-Tfes and X-Tfis are organized in operons that allow for their simultaneous expression. Among the X-Tfes of the phytopathogen Xanthomonas citri are Xac1918 and Xac0574. Xac1918 is carries a lysozyme superfamily domain, as well as a domain known as RTX (Repeats in Toxic) predict to bind calcium, while, Xac0574 has a domain belonging to the lipase 3 superfamily. Their possible inhibitors, Xac1917 e Xac0573 respectively, carry an N-terminal signal peptide containing a lipobox found in bacterial lipoproteins. The Xac0574 and Xac0573 proteins are both monomers in solution, They can form a stable 1:1 complex, that is thermodynamically favored (ΔG°= -12 Kcal/mol) with a dissociation constant of 2,45 nM. This affinity ensure that the bacterium is protected against the harmful effects of Xac0574 when it is produced intracellularly. We show that Xac0574 is a phospholipase A1, without lisophospholipase activity, and is able to hydrolyze the three most common phospholipids found in the membranes of Gram negative bacteria, namely phosphatidylglycerol (PG), cardiolipin and phosphatidylethanolamine (PE), presenting an apparent preference for PE. The enzymatic activity of Xac0574 explains the strong inhibition of growth of E. coli cells after its heterologous induction: a nearly 10-fold decrease in the cell population is observed when compared to the non-induced culture with the same construct. On the other hand, Xac0573 effectively inhibits the enzymatic activity of Xac0574. Furthermore, Xac0573 does not possess when forming the complex, besides not having phospholipase nor lysophospholipase activity.Crystals of Xac1918 and Xac0573 were produced which diffracted with to resolution of 3.0 and 2.5 Å, respectively. However, we were able to resolve the structure of only Xac0573. Xac0573 is composed of two anti-parallel sheet that form a ß-sandwich with three small helices. An alignment to Xac0573 homologs identified conserved regions at the ends of the protein that constitute two possible interfaces of interaction that may be responsible for blocking the access of the phospholipids to the catalytic site or impede the structural rearrangements of Xac0574 that are necessary for its enzymatic activity. Additionally, the topology of Xac0573 is similar to that to C2 domains, known in eukaryotes to bind lipids and calcium and to be involved in signaling processes mediated by lipid second messengers, membrane trafficking and membrane fusion mechanisms. Our results point to a new biological function of the C2 domain as an intracellular enzyme inhibitor in bacteria

Zero-inflated beta regression model for leaf citrus canker incidence in orange genotypes grafted onto different rootstocks / Modelo de regressão beta inflacionado de zeros para a incidência de cancro cítrico foliar em genótipos de laranja enxertados sobre diferentes porta-enxertos

Data with excess zeros are frequently found in practice, and the recommended analysis is to use models that adequately address the counting of zero observations. In this study, the Zero Inflated Beta Regression Model (BeZI) was used on experimental data to describe the mean incidence of leaf citrus canker in orange groves under the influence of genotype and rootstocks of origin. Based on the model, it was possible to quantify the odds that a null observation to mean incidence comes from a particular plant according to genotype and rootstock, and estimate its expected value according to this combination. Laranja Caipira rootstock proved to be the most resistant to leaf citrus canker as well as Limão Cravo proved to be the most fragile. The Ipiguá IAC, Arapongas, EEL and Olímpia genotypes have statistically equivalent chances.

Dados com excesso de zeros são encontrados muitas vezes na prática, e a análise recomendada é utilizar modelos que suportem adequadamente a contagem de observações nulas. Neste artigo, o Modelo de Regressão Beta Inflacionado de Zeros (BeZI) foi aplicado a dados experimentais para descrever a incidência média de cancro cítrico foliar em pomares de laranja sob a influência do genótipo e do porta-enxerto de origem. Com base no modelo, foi possível quantificar as chances de que uma observação nula para a incidência média seja proveniente de uma determinada planta, de acordo com o genótipo e o porta-enxerto, além de estimar o seu valor esperado conforme essa combinação. O porta-enxerto Laranja Caipira mostrou ser o mais resistente ao cancro cítrico foliar, assim como o Limão Cravo mostrou ser o mais suscetível. Os genótipos Ipiguá IAC, Arapongas, EEL e Olímpia apresentaram chances estatisticamente equivalentes.

Production of xanthanases by paenibacillus spp.: complete xanthan degradation and possible applications

Background: A number of microorganisms and their enzymes have been reported as xanthan depolymerizers. Paenibacillus species are well-known polysaccharide hydrolyzing bacteria. However, Paenibacillus alginolyticus and Paenibacillus sp. XD are the only species in the genus which are now known to degrade xanthan

Objectives: Complete biodegradation of the xanthan exopolysaccharide is a rarely found capability among microorganisms. The aim of this study is to survey xanthanase producing bacteria with an appropriate bioactivity for the biopolymer degradation under different environmental conditions

Materials and Methods: The bacteria were isolated based on viscosity reduction of the xanthan solution. Bacterial isolates were identified using rep-PCR [repetitive element-based genomic fingerprinting] and 16S rDNA sequencing. Xanthanases were characterized by measuring their activity at different temperatures, pH values, and NaCl concentrations. Degradation of other polysaccharides and xanthan degradation products were investigated based on the screening plate method and TLC [thin-layer chromatography], respectively

Results: Six isolates from different Paenibacillus species with a complete xanthan degrading capability were isolated from Urmia Lake. Phylogenetic analysis placed these strains within the genus Paenibacillus with the closest relatives that were found to be P. nanensis, P. phyllosphaerae, P. agaridevorans, P. agarexedens, and P. taohuashanense. These isolates displayed different levels of the xanthan biodegradation activity in temperatures ranging from 15 to 55 [degree]C and pH values from 4 to 11. Xanthanolytic activity was generally prevented in presence of NaCl [> 0.1 mol.L-1]. Furthermore, the isolated Paenibacillus spp. could degrade several other polysaccharides including xylan, CMC [carboxymethyl cellulose], starch, alginate, and pectin

Conclusion: Novel strains of the six different Paenibacillus species that were introduced in the present study are able to produce xanthanases with interesting characteristics. In light of the results from this study, special applications, particularly in healthcare, medicine, and the environment is hereby proposed for these enzymes

Estudos funcionais e bioquímicos sobre o reconhecimento e inibição de efetores de um sistema de secreção tipo IV de Xanthomonas citri subsp. citri. / Functional and biochemical son the recognition and Inhibition of effectors of a Type IV secretion System of Xanthomonas citri subsp. citri

Sistemas de Secreção Tipo IV (T4SSs), normalmente compostos por 12 proteínas (VirB1-VirB11 e VirD4) são tipicamente associados às funções de conjugação bacteriana e transferência de fatores de patogenicidade para células hospedeiras. Mas também, muitas espécies da ordem Xanthomonadales possuem um T4SS associado a matar bactérias. O modelo atual de morte de uma célula-alvo mediada pelo T4SS é baseado na secreção de toxinas denominadas XVIPs ("Xanthomonas VirD4 interacting proteins") ou X-Tfe (Xanthomonadaceae-T4SS effector) no qual cada XVIP/X-Tfe apresenta uma proteína de imunidade cognata denominada X-Tfi (Xanthomonadaceae-T4SS immunity protein). Demonstramos que um XVIP, XAC2609, é secretado através do T4SS de modo que depende de contato célula-célula e do seu domínio XVIPCD ("XVIP conserved domains"). A porção N-terminal de XAC2609 codifica um domínio GH19 que cliva a peptideoglicana de E. coli, mas perde a sua atividade na presença do seu inibidor cognato, o X-Tfi XAC2610. Portanto, XAC2609/XAC2610 formam um par de proteínas efetora/imunidade associado ao T4SS de X. citri. Através de diferentes técnicas de microscopias utilizando a cepa Δxac2610, foi observado que XAC2610 protege o envelope celular de X. citri contra efeitos de autólise celular promovidos pela atividade de XAC2609. Ensaios funcionais baseados nas observações de fenótipos de colônias e de formação de biofilme mostraram que XAC2610 confere imunidade para X. citri contra uma atividade 7 intrínseca de XAC2609. A proteína com o papel de reconhecer os substratos através da interação com os sinais de secreção do T4SS é VirD4. No T4SS de X. citri, existe a hipótese de que o domínio XVIPCD seja o sinal de secreção presente nas XVIPs. Logo, os aspectos bioquímicos e biofísicos da interação VirD4-XVIPCD foram investigados através de experimentos de co-purificação por cromatografia de afinidade e exclusão molecular, RMN e SAXS. Demonstramos que o domínio AAD de VirD4 (VirD4AAD) está associado a interagir especificamente com o domínio XVIPCD de XAC2609 (XAC2609XVIPCD), formando um heterodímero em solução. VirD4AAD é um domínio globular e monomérico e XAC2609XVIPCD é desenovelado mas se enovela concomitante à interação com VirD4AAD. Construções de XAC2609 contendo mutações pontuais no domínio XVIPCD foram utilizadas em ensaios in vivo de secreção pela X. citri e ensaios in vitro de interação com VirD4AAD por titulação monitorada por calorimetria isotérmica (ITC). Através desses experimentos, observamos que uma forte interação entre VirD4AAD-XAC2609XVIPCD é essencial para secreção de XAC2609 via o T4SS. Esses resultados permitem concluir que o domínio XVIPCD é o sinal de secreção dos substratos do T4SS de X. citri e que o AAD confere especificidade à VirD4 por interagir com o XVIPCD. Finalmente, através de ensaios de competições bacterianas entre E. coli e X. citri, foram observados diferentes fenótipos associados à função do T4SS: i) nocautes gênicos das subunidades estruturais VirB5, VirB11 abolem a função do T4SS em X. citri.; ii) nocautes de xac2611, apresentaram uma maior vantagem adaptativa do que a cepa selvagem de X. citri em competições e a expressão epissomal de XAC2611 inibe fortemente a função do T4SS e iii) a atividade ATPásica de VirD4 é essencial para a função do sistema e a expressão de mutantes 8 de VirD4 exerce um fenótipo de dominância negativa sobre a função do T4SS em X. citri

The Type IV secretion System (T4SS) is typically associated with the function of bacterial conjugation and as a pathogenicity factor. T4SSs are normally composed of 12 proteins, VirB1-VirB11 and VirD4. Many species of the order Xanthomonadales possess a T4SS associated with killing bacteria. The current model of the T4SS killing is based on the secretion of toxins denominated XVIPs/X-Tfes (Xanthomonas VirD4 interacting proteins) /(Xanthomonadaceae-T4SS effector) in which each XVIP/X-Tfe has a cognate immunity protein denominated X-Tfi (Xanthomonadaceae-T4SS immunity protein). We demonstrate that an XVIP, XAC2609, is secreted through the T4SS so that it depends on cell-cell contact and its XVIPCD domain ("XVIP conserved domains"). The N-terminal portion of XAC2609 encodes a GH19 domain which cleaves the E. coli peptidoglycan but loses its activity in the presence of its cognate inhibitor, X-Tfi XAC2610. Therefore, XAC2609 /XAC2610 form a pair of effector/immunity proteins associated with X. citri T4SS. By using the X. citri Δxac2610 strain, has been shown through different microscopic techniques that XAC2610 protects the cell envelope of X. citri against the effects of cellular autolysis promoted by XAC2609 activity. Functional assays based on observations of colony phenotypes and biofilm formation has shown that XAC2610 confers immunity to X. citri against an intrinsic activity of XAC2609. VirD4 is the protein that recognizes the substrates through the interaction with the T4SS secretion signals. In the T4SS of X. citri, is hypothesized that the XVIPCD domain is the secretion signal present in the XVIPs. Here, the biochemical and biophysical aspects of the VirD4-XVIPCD interaction were investigated through Pull- Down, Molecular Exclusion Chromatography, NMR and SAXS assays. It has been shown the AAD domain of VirD4 (VirD4AAD) is associated with specifically interacting with the XAC2609XVIPCD domain (XAC2609XVIPCD), forming a heterodimer in solution. VirD4AAD is a globular and monomeric domain while XAC2609XVIPCD is elongated, but upon interaction with VirD4AAD goes through structural compaction process. Constructs of XAC2609 containing point mutations in the XVIPCD domain were used to perform secretion experiments in X. citri and Isothermal titration calorimetry against VirD4AAD. Through these assays, it has been characterized that a strong interaction between VirD4AAD-XAC2609XVIPCD is essential for secretion of XAC2609 via T4SS. Consequently, these results allow concluding that the XVIPCD domain is the secretion signal of X. citri T4SS substrate and the AAD confer specificity to VirD4 by interact with the XVIPCD domains. Finally, bacterial competitions between E. coli and X. citri showed different phenotypes associated with T4SS function: i) virB5, virB11 knockouts abolish the function of T4SS in X. citri.; ii) knockouts of xac2611 exhibited a higher adaptive efficiency than the wild-type X. citri strain in competitions, but the expression of XAC2611 abolishes the function of T4SS in the wild strain of X. citri; iii) The ATPase activity of VirD4 is essential and exerts a negative dominance over the T4SS function in X.citri

A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

Chemical products induce resistance to Xanthomonas perforans in tomato

The bacterial spot of tomato, caused by Xanthomonas spp., is a very important disease, especially in the hot and humid periods of the year. The chemical control of the disease has not been very effective for a number of reasons. This study aimed to evaluate, under greenhouse conditions, the efficacy of leaf-spraying chemicals (acibenzolar-S-methyl (ASM) (0.025 g.L⁻¹), fluazinam (0.25 g.L⁻¹), pyraclostrobin (0.08 g.L⁻¹), pyraclostrobin + methiran (0.02 g.L⁻¹ + 2.2 g.L⁻¹), copper oxychloride (1.50 g.L⁻¹), mancozeb + copper oxychloride (0.88 g.L⁻¹ + 0.60 g.L⁻¹), and oxytetracycline (0.40 g.L⁻¹)) on control of bacterial spot. Tomatoes Santa Clara and Gisele cultivars were pulverized 3 days before inoculation with Xanthomonas perforans. The production of enzymes associated with resistance induction (peroxidase, polyphenol oxidase, phenylalanine ammonia-lyase, β-1,3-glucanase, and protease) was quantified from leaf samples collected 24 hours before and 24 hours after chemical spraying and at 1, 2, 4, 6, and 8 days after bacterial inoculation. All products tested controlled bacterial spot, but only ASM, pyraclostrobin, and pyraclostrobin + metiram increased the production of peroxidase in the leaves of the two tomato cultivars, and increased the production of polyphenol oxidase and β-1,3-glucanase in the Santa Clara cultivar.

Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato

Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding beta-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction.

Controle químico da mancha bacteriana em mudas de tomate para processamento industrial / Chemical control of bacterial spot in seedlings of tomato for industrial processing

Com o objetivo de avaliar opções de produtos para o controle da mancha bacteriana em mudas de tomate, foram realizados dois ensaios independentes, em viveiro comercial, no município de Rio Verde ­ GO, nos períodos de novembro a dezembro de 2009. Os experimentos foram conduzidos em delineamento experimental inteiramente casualizado com três repetições, utilizando o híbrido Heinz 9553. A parcela foi representada por 450 mudas em uma bandeja comercial. O primeiro ensaio consistiu nos tratamentos: 1 - testemunha; 2 - oxicloreto de cobre; 3 - hidróxido de cobre; 4 - acibenzolar-S-metil; 5 - metiram + piraclostrobina; 6 - famoxadona + mancozebe; 7 - cloreto de benzalcônio. O segundo ensaio consistiu nos mesmos tratamentos do primeiro ensaio acrescidos da aplicação do regulador de crescimento - paclobutrazol. As aplicações foram realizadas via pulverização foliar, utilizando pulverizador costal de barra com pressão constante. Após 29 dias da semeadura, o isolado EH 2008-13 de X. perforans, foi inoculado por meio da imersão das mudas em bandeja. A severidade da mancha bacteriana foi avaliada em 15 folíolos de cada parcela aos 16 dias após a inoculação. Não houve diferenças significativas no primeiro ensaio, mas detectou-se diferença significativa no segundo onde houve a aplicação do regulador de crescimento (P= 0,001). Os tratamentos acibenzolar-S-metil e famoxadona + mancozebe apresentaram valores médios de severidade inferiores à testemunha, no entanto, não diferiram significativamente dela. O tratamento metiram + piraclostrobina apresentou maior severidade da mancha bacteriana, não demonstrando ser eficaz no controle da doença em mudas nas condições testadas neste ensaio. Os resultados indicam possível efeito do regulador de crescimento sobre a ação dos produtos testados.

With the aim of evaluate chemical options to the control of bacterial spot in tomato seedlings, two independent experiments were carried out at a commercial tomato nursery in Rio Verde, Goiás, during the periods of November to December of 2009. The hybrid Heinz 9553 was used. The trials were in a completely randomized design with three replications. The plots were represented by a commercial plantlet trays of 450 cells. The treatments were: 1 - control; 2 - copper oxicloreto; 3 - copper hydroxide; 4 - acibenzolar-S-methyl; 5 - metiram + pyraclostrobin, 6 - famoxadone + mancozebe ; 7 ­ benzalkonium chloride. The experiments differed as for the paclobutrazol application, in the second experiment. The products were applied by using a CO2 portable sprayer with constant pressure. The inoculation occurred at 29 days after sowing, with isolated EH 2008-13 of Xanthomonas perforans which was originated from Rio Verde. The aerial part of the plantlets were immersed during 1 minute in a 18 L of the bacterial suspension placed in a large plastic tray. Disease severity was evaluated on 15 leaflets per plot at 16 days after inoculation. It was expressed in terms of average percentage of foliar area with symptoms by using the computational program Quant 2002. For the experiment without use of growth regulator there were not significant differences among the treatments. Despite presenting significant differences, in the for the paclobutrazol application, except for treatment metiram + pyraclostrobin, which resulted in the higher disease value, all treatments were not significantly different from the check-control. In that trial, acibenzolar-S-methyl following by famoxadone + mancozebe, were the only ones that presented inferior values comparing with the check-control, however not being significantly different. It can be inferred that interaction between the growth regulator and some treatments should exist.

Tripanocide and antibacterial activity ofAlvaradoa subovata Cronquist extracts / Actividad tripanocida y antibacteriana de extractos de Alvaradoa subovata Cronquist

We studied antioxidant, antibacterial and tripanocide activities of Alvaradoa subovata extracts. The ethanolic extracts showed the greatest DPPH radical scavenging capacity, especially that of bark with an IC50 = 4.7 +/- 0.18 ug/mL. Wood dichloromethane extract displayed growth inhibition of the phytopathogenic bacteria Xanthomona axonopodis in the disk diffusion assay and showed a MIC value of 100 ug/ml. It also showed growth inhibition of Trypanosoma cruzi (IC50 = 0.063 +/- 0.003 mg/mL). A fraction of this extract, which has emodin as the main component, showed tripanocide activity (60 percent of growth inhibition at 100 ug/mL). The main compounds in wood dichloromethane extract were anthraquinones, identified as chrysophanol and emodin, and coumarins, of which scopoletin was identified. These three compound s could serve as analytical markers of the extract. The results of this study show that wood extract of A. subovata constitute a source of bioactive compounds such as antiparasitic and pesticides agents.

En el presente trabajo se estudió la actividad antioxidante, antibacteriana y tripanocida de extractos de Alvaradoa subovata. La mayor actividad depuradora de radicales libres se observó en el extracto etanólico de corteza (CI50 = 4.7 +/- 0.18 ug/mL). El extracto en diclorometano de madera inhibió el crecimiento de la bacteria fitopatógena Xanthomona axonopodis con una CIM = 100 ug/mL. El mismo extracto mostró inhibición del crecimiento de Trypanosoma cruzi (CI50 = 0.063 +/- 0.003 mg/mL). Una fracción de este extracto (100 ug/mL), cuyo componente mayoritario es emodina, inhibió en un 60 por ciento el crecimiento del parásito. Los compuestos mayoritarios detectados en el extracto de madera fueron antraquinonas, entre las cuales se identificaron emodina y crisofanol, y la cumarina escopoletina. Estos tres compuestos podrían servir como marcadores analíticos del extracto. Los resultados de este trabajo muestran que los extractos de A. subovata constituyen una fuente de compuestos bioactivos con potencial como antiparasitarios y plaguicidas.