A disease predictive model based on epidemiological factors for the management of bacterial leaf blight of rice / Um modelo preditivo de doenças baseado em fatores epidemiológicos para o manejo da queima bacteriana das folhas do arroz

Rice is a widely consumed staple food for a large part of the world's human population. Approximately 90% of the world's rice is grown in Asian continent and constitutes a staple food for 2.7 billion people worldwide. Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae is one of the devastating diseases of rice. A field experiment was conducted during the year 2016 and 2017 to investigate the influence of different meteorological parameters on BLB development as well as the computation of a predictive model to forecast the disease well ahead of its appearance in the field. The seasonal dataset of disease incidence and environmental factors was used to assess five rice varieties/ cultivars (Basmati-2000, KSK-434, KSK-133, Super Basmati, and IRRI-9). The accumulated effect of two year environmental data; maximum and minimum temperature, relative humidity, wind speed, and rainfall, was studied and correlated with disease incidence. Average temperature (maximum & minimum) showed a negative significant correlation with BLB disease and all other variables; relative humidity, rainfall, and wind speed had a positive correlation with BLB disease development on individual varieties. Stepwise regression analysis was performed to indicate potentially useful predictor variables and to rule out incompetent parameters. Environmental data from the growing seasons of July to October 2016 and 2017 revealed that, with the exception of the lowest temperature, all environmental factors contributed to disease development throughout the cropping season. A disease prediction multiple regression model was developed based on two-year data (Y = 214.3-3.691 Max T-0.508 Min T + 0.767 RH + 2.521 RF + 5.740 WS), which explained 95% variability. This disease prediction model will not only help farmers in early detection and timely management of bacterial leaf blight disease of rice but may also help reduce input costs and improve product quality and quantity. The model will be both farmer and environmentally friendly.

O arroz é um alimento básico amplamente consumido por grande parte da população humana mundial. Aproximadamente 90% do arroz do mundo é cultivado no continente asiático e constitui um alimento básico para 2,7 bilhões de pessoas em todo o mundo. O crestamento bacteriano das folhas (BLB) causado por Xanthomonas oryzae pv. oryzae é uma das doenças devastadoras do arroz. Um experimento de campo foi realizado durante os anos de 2016 e 2017 para investigar a influência de diferentes parâmetros meteorológicos no desenvolvimento do BLB, bem como o cálculo de um modelo preditivo para prever a doença bem antes de seu aparecimento em campo. O conjunto de dados sazonais de incidência de doenças e fatores ambientais foi usado para avaliar cinco variedades/cultivares de arroz (Basmati-2000, KSK-434, KSK-133, Super Basmati e IRRI-9). O efeito acumulado de dados ambientais de dois anos; temperatura máxima e mínima, umidade relativa do ar, velocidade do vento e precipitação pluviométrica foram estudados e correlacionados com a incidência da doença. A temperatura média (máxima e mínima) apresentou correlação significativa negativa com a doença BLB e todas as outras variáveis; umidade relativa, precipitação e velocidade do vento tiveram uma correlação positiva com o desenvolvimento da doença BLB em variedades individuais. A análise de regressão stepwise foi realizada para indicar variáveis preditoras potencialmente úteis e para descartar parâmetros incompetentes. Os dados ambientais das safras de julho a outubro de 2016 e 2017 revelaram que, com exceção da temperatura mais baixa, todos os fatores ambientais contribuíram para o desenvolvimento da doença ao longo da safra. Um modelo de regressão múltipla de previsão de doença foi desenvolvido com base em dados de dois anos (Y = 214,3-3,691 Max T-0,508 Min T + 0,767 RH + 2,521 RF + 5,740 WS), que explicou 95% de variabilidade. Este modelo de previsão de doenças não só ajudará os agricultores na detecção precoce e gestão atempada da doença bacteriana das folhas do arroz, mas também pode ajudar a reduzir os custos de insumos e melhorar a qualidade e a quantidade do produto. O modelo será agricultor e ambientalmente amigável.

The role of iron-uptake factor PiuB in pathogenicity of soybean pathogen Xanthomonas axonopodis pv. glycines / 生物工程学报

Iron is an essential element for living organisms that plays critical roles in the process of bacterial growth and metabolism. However, it remains to be elucidated whether piuB encoding iron-uptake factor is involved in iron uptake and pathogenicity of Xanthomonas axonopodis pv. glycines (Xag). To investigate the function of piuB, we firstly generated a piuB deletion mutant (ΔpiuB) by homologous recombination. Compared with the wild-type, the piuB mutant exhibited significantly reduced growth and virulence in host soybean. The mutant displayed markedly increased siderophore secretory volume, and its sensitivity to Fe3+, Cu2+, Zn2+ and Mn2+ was significantly enhanced. Additionally, the H2O2 resistance, exopolysaccharide yield, biofilm formation, and cell mobility of ΔpiuB were significantly diminished compared to that of the wild-type. The addition of exogenous Fe3+ cannot effectively restore the above characteristics of ΔpiuB. However, expressing piuB in trans rescued the properties lost by ΔpiuB to the levels in the wild-type. Taken together, our results demonstrated that PiuB is a potential factor for Xag to assimilate Fe3+, and is necessary for Xag to be pathogenic in host soybean.

A disease predictive model based on epidemiological factors for the management of bacterial leaf blight of rice

Abstract Rice is a widely consumed staple food for a large part of the worlds human population. Approximately 90% of the worlds rice is grown in Asian continent and constitutes a staple food for 2.7 billion people worldwide. Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae is one of the devastating diseases of rice. A field experiment was conducted during the year 2016 and 2017 to investigate the influence of different meteorological parameters on BLB development as well as the computation of a predictive model to forecast the disease well ahead of its appearance in the field. The seasonal dataset of disease incidence and environmental factors was used to assess five rice varieties/ cultivars (Basmati-2000, KSK-434, KSK-133, Super Basmati, and IRRI-9). The accumulated effect of two year environmental data; maximum and minimum temperature, relative humidity, wind speed, and rainfall, was studied and correlated with disease incidence. Average temperature (maximum & minimum) showed a negative significant correlation with BLB disease and all other variables; relative humidity, rainfall, and wind speed had a positive correlation with BLB disease development on individual varieties. Stepwise regression analysis was performed to indicate potentially useful predictor variables and to rule out incompetent parameters. Environmental data from the growing seasons of July to October 2016 and 2017 revealed that, with the exception of the lowest temperature, all environmental factors contributed to disease development throughout the cropping season. A disease prediction multiple regression model was developed based on two-year data (Y = 214.3-3.691 Max T-0.508 Min T + 0.767 RH + 2.521 RF + 5.740 WS), which explained 95% variability. This disease prediction model will not only help farmers in early detection and timely management of bacterial leaf blight disease of rice but may also help reduce input costs and improve product quality and quantity. The model will be both farmer and environmentally friendly.

Resumo O arroz é um alimento básico amplamente consumido por grande parte da população humana mundial. Aproximadamente 90% do arroz do mundo é cultivado no continente asiático e constitui um alimento básico para 2,7 bilhões de pessoas em todo o mundo. O crestamento bacteriano das folhas (BLB) causado por Xanthomonas oryzae pv. oryzae é uma das doenças devastadoras do arroz. Um experimento de campo foi realizado durante os anos de 2016 e 2017 para investigar a influência de diferentes parâmetros meteorológicos no desenvolvimento do BLB, bem como o cálculo de um modelo preditivo para prever a doença bem antes de seu aparecimento em campo. O conjunto de dados sazonais de incidência de doenças e fatores ambientais foi usado para avaliar cinco variedades/cultivares de arroz (Basmati-2000, KSK-434, KSK-133, Super Basmati e IRRI-9). O efeito acumulado de dados ambientais de dois anos; temperatura máxima e mínima, umidade relativa do ar, velocidade do vento e precipitação pluviométrica foram estudados e correlacionados com a incidência da doença. A temperatura média (máxima e mínima) apresentou correlação significativa negativa com a doença BLB e todas as outras variáveis; umidade relativa, precipitação e velocidade do vento tiveram uma correlação positiva com o desenvolvimento da doença BLB em variedades individuais. A análise de regressão stepwise foi realizada para indicar variáveis preditoras potencialmente úteis e para descartar parâmetros incompetentes. Os dados ambientais das safras de julho a outubro de 2016 e 2017 revelaram que, com exceção da temperatura mais baixa, todos os fatores ambientais contribuíram para o desenvolvimento da doença ao longo da safra. Um modelo de regressão múltipla de previsão de doença foi desenvolvido com base em dados de dois anos (Y = 214,3-3,691 Max T-0,508 Min T + 0,767 RH + 2,521 RF + 5,740 WS), que explicou 95% de variabilidade. Este modelo de previsão de doenças não só ajudará os agricultores na detecção precoce e gestão atempada da doença bacteriana das folhas do arroz, mas também pode ajudar a reduzir os custos de insumos e melhorar a qualidade e a quantidade do produto. O modelo será agricultor e ambientalmente amigável.

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).

Studies on the enzymatic specificity of diguanylate cyclases domains and the production of new second messengers / Estudos na especificidade enzimática de diguanilato ciclases e na produção de novos segundos mensageiros

In the first chapter, studies on substrate recognition and enzymatic activity of GGDEF domains are presented. Many proteins containing GGDEF domains are diguanylate cyclases (DGCs, EC 2.7.7.65), enzymes that catalyze the conversion of 2 GTP molecules into the second messenger c-di-GMP in prokaryotes. This molecule is primarily implicated in the transition between motile and sessile lifestyles, as well several other phenotypes. Redundancy and diversity of GGDEF domain sequences in many bacterial genomes raises the possibility that other enzymatic functions may yet be discovered. To test this hypothesis, i) the effect of point mutations on the structure and enzymatic activity of GGDEF domains is analyzed, ii) the enzymatic specificity of wild-type GGDEF domains from different proteins is also tested, and iii) when non-canonical products are detected, enzymatic models are studied to understand its preferential production. The principal results obtained from these studies are as follows. Seven mutants of the DGC PleD (a GGDEF containing-protein from Caulobacter crescentus) were constructed and the crystallographic structure of two of them was solved, showing that they are unlikely to bind the guanine moiety in its active site. Additionally, five mutants of XAC0610, another DGC from Xanthomonas citr, were constructed and their substrate specificities were evaluated. None of those mutants were able to use ATP as a substrate. Finally, seven different GGDEF domain-containing DGCs from different sources were expressed and purified and their enzymatic specificities were tested with several nucleotide triphosphates. One enzyme, GSU1658 from Geobacter sulfurreducens was particularly promiscuous and shown to produce c-di-GMP, c-di-AMP, c-di-IMP, c-di-2´dGMP, cGAMP, c-GIMP, and c-AIMP. Interestingly, XAC0610 was able to recognize 2´dGTP as substrate. Analysis of enzyme kinetics of XAC0610 in presence of 2´dGTP and/or GTP showed the preferential formation of the hybrid linear product pppGp2´dG. The second chapter present studies on cyanide metabolism in Bacillus with focus on the cyanide dihydratase of Bacillus safensis. Cyanide is widely used in industries due to its high affinity for metals. This same ability confers potent toxicity to this compound. Thus, industries must reduce the cyanide concentration from wastewater before its final disposal. Physical, chemical, and biological methods have been developed to achieve this goal, but knowledge about metabolic pathways and the biology of enzymes involved in cyanide degradation is still scarce. Here, the isolation of a Bacillus safensis strain from mine tailings in Peru is described. Classification of this strain was done through a comparative analysis of 132 core genomes of strains from the Bacillus pumilus group. Sequence analysis determined that a cyanide dihydratase (CynD, EC 3.5.5.1)) encoded in the genome of the isolated strain was likely the enzyme responsible for cyanide degradation. Confirmation of the cyanide degrading activity of CynD from this strain was achieved by cloning, expression and purification of the enzyme and its enzymatic characterization. CynD from this strain was active up to pH 9 and oligomerization patterns analyzed by SEC-MALS and electron microscopy showed that the enzyme forms large helical structures at pH 8 and smaller structures at higher pHs. Finally, we show that CynD expression is strongly induced in the presence of cyanide. The last two years of graduate studies were carried out in the context of the COVID-19 pandemic. Thanks to the large amount of publicly available genomic data, we were able to carry out studies on the worldwide dynamics of the spread of SARS-CoV-2 mutants forms. In the first year of the pandemic, genomic classification of 171,461 genomes showed the presence of five major haplotypes based on nine mutations. The worldwide distribution and the temporal evolution of frequency of these haplotypes was carefully analyzed. All the haplotypes were identified in the six regions analyzed (South America, North America, Europe, Asia, Africa, and Oceania); however, the frequency of each of them was different in each of these regions. As of September 30, 2020, haplotype 3 (or operational taxonomic unit 3, OTU_3) was the most prevalent in four regions (South America, Asia, Africa, and Oceania). OTU_5 was the most prevalent in North America and OTU_2 in Europe. Temporal dynamics of the haplotypes showed that OTU_1 became nearly extinct after 8 months of pandemic (November 2020). Other OTUs are still present in different frequencies all around the world, while currently generating new variants. Based on their temporal dynamics, a classification scheme of 115 SARS-CoV-2 mutations identified from 1,058,020 SARS-COV-2 genomes was also performed. Three types of temporal dynamics of mutations were identified: i) High-Frequency mutations are characterized by a rapid increase in frequency upon its appearance, ii) medium and iii) low-frequency mutations maintain mid or low-frequencies for several months and can be region-specific. Finally, we performed a correlation analysis of the effective reproduction number (Rt) of SARS-CoV-2 harboring the high-frequency mutation N501Y with the level of control measures adopted in specific jurisdictions. We show that Rt is negatively correlated with the level of control measures in eight of the nine countries analyzed. This negative correlation was similar when we analyzed the Rt of SARS-CoV-2 not-harboring N501Y. Thus, the control measures likely diminish the Rt of both SARSCoV-2 wild-type and N501Y

O presente trabalho está dividido em três capítulos sobre linhas de pesquisa diferentes desenvolvidas pelo autor durante o período de doutorado No primeiro capítulo, são apresentados estudos relacionados ao reconhecimento estrutural de substratos e análise enzimática de domínios GGDEF com atividade diguanilato ciclase (EC 2.7.7.65). As proteínas contendo domínios GGDEF estão relacionados à produção enzimática do segundo mensageiro c-di-GMP, a partir de duas moléculas de GTP, em procariotos. Esta molécula está principalmente envolvida na transição entre os estilos de vida móveis e sésseis, bem como vários outros fenótipos. Redundância e diversidade de sequências de domínio GGDEF aumentam a possibilidade de que outras funções enzimáticas ainda possam ser descobertas. Para testar esta hipótese, i) o efeito de mutações pontuais na estrutura e atividade enzimática dos domínios GGDEF é analisado, ii) a especificidade enzimática de domínios GGDEF de enzimas diferentes também é testada e iii) quando produtos não canônicos são detectados, modelos enzimáticos são estudados para entender sua produção preferencial. Como resultados mais importantes, sete mutantes do PleD (uma proteína contendo GGDEF) foram construídos e a estrutura cristalográfica de dois delas foi resolvida, mostrando que é improvável que eles liguem à porção guanina em seu sítio ativo. Além disso, cinco mutantes da proteína XAC0610 de Xanthomonas citri foram construídos e sua capacidade de usar ATP ou GTP como substrato foi avaliada. Nenhum desses mutantes foi capaz de usar ATP como substrato. Finalmente, sete outras proteínas contendo GGDEF foram purificadas e sua especificidade enzimática foi avaliada com vários trifosfatos de nucleotídeos. Uma enzima promíscua chamada GSU1658 mostrou produzir c-di-GMP, c-di-AMP, c-di-IMP, c-di-2´dGMP, c-GAMP, cGIMP e c-AIMP. Curiosamente, o XAC0610 foi capaz de reconhecer 2´dGTP como substrato. A análise da cinética enzimática de XAC0610 na presença de 2´dGTP e GTP mostrou a formação preferencial do produto linear híbrido pppGp2´dG. O segundo capítulo aborda estudos sobre o metabolismo do cianeto em Bacillus com foco na cianeto dihidratase de Bacillus safensis. O cianeto é amplamente utilizado nas indústrias devido à sua alta afinidade com os metais. Esta mesma capacidade confere toxicidade potente a este composto. Assim, as indústrias têm que reduzir a concentração de cianeto das águas residuais antes de sua disposição final. Métodos físicos, químicos e biológicos têm sido desenvolvidos para atingir esse objetivo, mas o conhecimento sobre as vias metabólicas e a biologia das enzimas envolvidas na degradação do cianeto ainda é escasso. Aqui, é descrito o isolamento de uma cepa de Bacillus safensis de rejeitos de minas no Peru. A classificação desta cepa foi feita através de uma análise comparativa de 132 core genomes de cepas do grupo de Bacillus pumilus. Em seguida, determinamos que uma cianeto dihidratase (CynD, EC 3.5.5.1) codificada no genoma da cepa isolada era provavelmente a enzima responsável pela degradação do cianeto. A confirmação da atividade degradante de cianeto de CynD desta cepa foi feita por clonagem, expressão e purificação da enzima e realização de caracterização enzimática. O CynD desta cepa é ativo até pH 9 e os padrões de oligomerização analisados por SEC-MALS mostraram que a enzima forma longas estruturas helicoidais em pH 8 e estruturas menores enquanto o pH aumenta. Finalmente, foi demonstrado que a expressão de CynD é fortemente induzida na presença de cianeto. Os últimos dois anos do doutorado foram realizados no contexto da pandemia COVID- 19. Vários laboratórios se dedicaram a gerar conhecimento para ajudar no combate à pandemia. Nesta situação e graças à grande quantidade de dados genômicos disponíveis publicamente, estudos sobre a dinâmica das mutações do SARS-CoV-2 foram realizados. No primeiro ano da pandemia, a classificação genômica de 171.461 genomas mostrou a presença de cinco haplótipos principais com base em nove mutações. A distribuição mundial e a mudança de frequência desses haplótipos foram analisadas cuidadosamente. Todos os haplótipos foram identificados nas seis regiões analisadas (América do Sul, América do Norte, Europa, Ásia, África e Oceania); no entanto, a frequência de cada um deles foi diferente em cada uma dessas regiões. Em 30 de setembro de 2020, o haplótipo 3 (ou unidade taxonômica operacional 3, OTU_3) era o mais prevalente em quatro regiões (América do Sul, Ásia, África e Oceania). OTU_5 foi o mais prevalente na América do Norte e OTU_2 na Europa. A dinâmica temporal dos haplótipos mostrou que OTU_1 parece perto da extinção após 8 meses de pandemia (novembro de 2020). Outros OTUs ainda estão presentes em diferentes frequências em todo o mundo, mesmo atualmente gerando novas variantes. Com base em sua dinâmica temporal, um esquema de classificação de 115 mutações SARS-CoV-2 identificadas a partir de 1.058.020 genomas SARS-COV-2 também foi feito. Três tipos de dinâmica temporal de mutações foram identificados: i) Mutações de alta frequência, ii) mutações de média frequência e iii) mutações de baixa frequência. Finalmente, foi analisada a correlação do número de reprodução efetiva (Rt) do SARS-CoV-2 que contém a mutação de alta frequência N501Y com o nível de medidas de controle, mostrando que seu Rt está negativamente correlacionado com o nível de medidas de controle em oito dos nove países analisados. Esta correlação negativa foi semelhante quando foi analisado o Rt de SARS-CoV-2 sem a mutação N501Y. Assim, as medidas de controle provavelmente diminuirão o Rt de SARS-CoV-2 tipo selvagem e N501Y

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.

Assessment of resistance of hybrids and lineages of passion fruit to bacteriosis under protected cultivation / Avaliação da resistência de híbridos e linhagens de maracujazeiro à bacteriose sob cultivo protegido

Yield and longevity of yellow passion fruit have been reduced by diseases such as the bacterial spot caused by Xanthomonas axonopodis pv. passiflorae. Genetic resistance has been confirmed as the most efficient and economical correct option to minimize this disease problem. Aiming at it, the objective of this research was to evaluate the incidence, severity and progression of the disease in 12 genotypes of sour passion fruit, in seedling stage in nursery greenhouse after inoculation of Xanthomonas axonopodis pv. passiflorae. The inoculation was performed with an isolate collected in the Pipiripau Rural Nucleus, Brasilia-DF, named UnB-1397 (1x106 CFU/mL), through induction of injuries. There were performed four assessments, with interval of 7 days except the first which was performed 11 days after inoculation. The incidence was estimated by the percentage of plants affected. To evaluate the severity, it was used the diagrammatic scale validated by Costa et al. (2018), with adaptations, using the measurement of the affected area by necrotic lesions on the leaf. All genotypes were susceptible to bacteriosis, 5 being considered moderately susceptible: F1BRS Pérola do Cerrado x Rosa Intenso, Mar20#21, Mar20#15b, Mar20#24xMar20#40 and FB200PL4R2 x Mar20#2005, with a mean of severity ranging from 11 to 25% of injured area in leaves.

A produtividade e a longevidade dos pomares de maracujazeiro-azedo têm sido comprometidas em razão de doenças como a bacteriose, causada por Xanthomonas axonopodis pv. passiflorae. A resistência genética tem se confirmado como a opção mais eficiente e econômica para minimizar tal problema. Dessa forma, o objetivo deste trabalho foi avaliar a reação de 12 genótipos de maracujazeiro, em fase de mudas, sob cultivo protegido, à Xanthomonas axonopodis pv. passiflorae. A inoculação com isolado denominado UnB-1397 (1x106 CFU/mL),coletado no Núcleo Rural de Pipiripau, Brasília-DF, se deu pela indução de ferimentos. Foram realizadas 4 avaliações, com intervalo de 7 dias, sendo a primeira avaliação realizada 11 dias após a inoculação. A incidência foi estimada pela porcentagem de plantas afetadas. Para avaliação da severidade, foi utilizada escala diagramática validada por Costa et al. (2018), com adaptações, utilizando-se a mensuração da área foliar atingida por lesões nas folhas. Todos os genótipos se mostraram suscetíveis à bacteriose, sendo 5 considerados moderadamente suscetíveis: F1 BRS Pérola do Cerrado x Rosa Intenso, Mar20#21, Mar20#15b, Mar20#24 x Mar20#40 e FB200PL4R2 x Mar20#2005, apresentando uma média de severidade que variou de 11 a 25% de área ou tecidos foliares lesionados.

Genomewide analysis of the CIII peroxidase family in sweet orange (Citrus sinensis) and expression profiles induced by Xanthomonas citri subsp. citri and hormones

Class III peroxidase (CIII prx) is a plant-specific multigene family that regulates the physiological and stress responses. This research aimed to exhaustively annotate and analyse the CIII prx family in sweet orange and to explore the regulated expression profiles by Xanthomonas citri subsp. citri (Xcc) and plant hormones. We further assessed the relationship between CIII prxs and citrus bacterial canker. The phylogeny, gene structure, conserved motifs, gene duplications and microsynteny of the CIII prx family were analysed. Expression profiles of specific CsPrxs induced by Xanthomonas citri subsp. citri and plant hormones were detected by quantitative reverse transcription-polymerase chain reaction. Subcellular localization was analysed through transient expression assessments. A total of 72 CIII prx members were identified from the genomes of sweet orange. In all chromosomes of sweet orange, the CsPrxs could be detected except in chromosome 8. In addition, three segmental duplications, four tandem duplications and 11 whole-genome duplications occurred among the CsPrxs, contributing to the family size expansion. From the Ka/Ks ratios, 15 of 18 duplicated CsPrxs pairs have experienced purifying selection process. A total of 15 conserved motifs were detected in CsPrxs, four of which were detected in all complete CsPrxs. A total of 12 expressed genes were identified from the EST database. The expression trends of 12 CsPrxs were differently expressed at different stages of infection by Xcc, five of which were potential candidate genes involved in Xcc resistance. These genes could be induced by salicylic acid and methyl jasmonate, and were extracellular proteins. These results further support our understanding of CIII prxs in citrus, particularly in citrus bacterial canker studies

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.

Characterization of Xanthomonas campestris pv. viticola causing bacterial leaf spot of grapes in Maharashtra, India

Aim: The aim of the present study was to isolate, characterize and identify pathogenic bacteria from bacterial leaf spot infected grape vineyards of Maharashtra. Methodology: Collected diseased samples were subjected to isolation. All isolates were further subjected to different morphological and biochemical characterization. These isolates were also used to standardize inoculation methods to study host range and test pathogenicity to confirm infectivity. Genomic DNA was isolated from bacteria for phylogeneitc analysis using 16 rDNA. Results: The disease is characterized by water soaked, angular leaf spot which later turns to irregular, dark brown to black necrotic region on the leaf surface. Microbial studies confirmed that it is a Gram negative, rod shaped bacterium with white, mucoid, glistening and convex circular colonies on artificial medium. Pathogenicity test was performed to confirm virulence. Phylogenetic analysis of 16S rDNA gene exhibited more than 99% similarity with other Xanthomonas campestris pv. viticola. The bacterium was found resistant to vancomycin, peniciliin and oxacillin antibiotics. The bacterium is also capable of infecting Mangifera indica and Citrus limon along with eight weed species.

In vitro Analysis of Antimicrobial Susceptibility of Xanthomonas axonopodis pv. punicae and Comparative Study of Protein Profiling under Biotic Stress

Bacterial blight of pomegranate caused by X. axonopodis pv. Punicae (XAP) assumed epidemic form and resulted in economic burden on farmers. In the current study the pathogen infected samples were collected and the isolated XAP was identity and confirmed through the morphological, biochemical characterization and Pathogenicity test. Bacterium was reisolated from infected plant to prove Koch’s postulates. Efficacy of different chemicals and oils were tested by disc diffusion assay and turbidometrically. Bronopol 3000 ppm (25.6±1.6 mm) and Clove oil (18.0±0.7 mm) formed highest zone of inhibition Turbidometri showed the highest O.D. (0.908 nm) by Copper oxy chloride and Neem oil showed maximum inhibition of growth with O.D. (0.842 nm). Biotic stress (pathogen) induced protein response was studies by using SDS-PAGE method after protein extraction from XAP, healthy P. granatum L. and infected P. granatum L. The protein band pattern showed the unique band no. 2 (Mol.Wt.66000 Da) in infected P. granatum L. as compared to the banding pattern of XAP and healthy P. granatum L. The over expressed protein due to biotic stress could be useful as a marker for detection of the disease at the early stage and for control of the diseases after knowing the biochemical significance of the protein.

The STK domain of the cassava bacterial blight resistance protein RXam1 interacts with an E3 Ubiquitin Ligase / El dominio stk de la proteína de resistencia a la bacteriosis vascular de yuca rxam1 interactúa con una e3 ubiquitin ligasa

RESUMEN La yuca (Manihot esculenta) representa el pilar de la seguridad alimentaria para cerca de mil millones de personas, principalmente en las zonas tropicales. Uno de los factores limitantes de la producción de yuca es la bacteriosis vascular causada por la bacteria Xanthomonasaxonopodis pv. manihotis (Xam). Recientemente se identificó el gen RXaml el cual confiere resistencia parcial de yuca a cepas de Xam. RXaml codifica una proteína con un dominio LRR (Leucine Rich Repeats) extracelular y un dominio STK (Serina Treonina Kinasa) citoplasmático; estas proteínas son conocidas como RLKs (Receptor Like Kinases). En este estudio se realizó el tamizaje de una librería de ADNc de yuca mediante doble híbrido de levadura para identificar las posibles proteínas que interactúan con el dominio STK de RXam1. El tamizaje de 3x108 clones permitió identificar y confirmar cinco clones de ellos los cuales corresponden al mismo gen, el cual codifica para una proteína que presenta un dominio central de dedos de zinc CHY, seguido por un dominio C-terminal "RING finger" y un "Zinc ribbon" el cual fue denominado CRFE3-1 (Cassava RING Finger E3 ligase). La interacción entre STK y CRFE3-1 fue altamente especifica ya que se demostró también por doble híbrido que STK no interactúa con una E3 ligasa de Arabidopsis, altamente similar a CRFE3-1, así como tampoco CRFE3-1 interactúa con el dominio STK de un RLK de lechuga similar a RXam1. La identificación de CRFE3-1 sugiere que mecanismos de degradación proteica son importantes para regular la actividad de RXam1.

ABSTRACT Cassava (Manihot esculenta) represents food security support for nearly one billion people, mainly in the tropics. One of the limiting factors of cassava's production is cassava bacterial blight, caused by the bacterium Xanthomonas axonopodis pv. manihotis (Xam). Recently, the RXam1 gene was identified, which confers partial resistance to some Xam strains. RXam1 encodes a protein with an extracellular LRR (Leucine Rich Repeats) domain and a cytoplasmic STK (Serine Threonine Kinase) domain; these proteins are known as RLK (Receptor-like Kinases). In this study, a cassava cDNA library was screened using a yeast Two-hybrid assay to identify possible proteins interacting with the STK domain of RXam1. Screening of 3x108 clones allowed identifying and confirming five of them, which correspond to the same gene, and code for a protein that has a core domain of zinc fingers CHY, followed by a C-terminal "RING finger" domain and a "Zinc ribbon". This gene was called CRFE3-1 (Cassava RING Finger E3 ligase). It was also demonstrated by yeast Two-hybrid that STK does not interact with an E3 ligase of Arabidopsis that is highly like CRFE3-1. CRFE3-1 did not show interaction with the STK domain of an RLK of lettuce related to RXam1, indicating a highly specific interaction between cassava RXam1 STK and CRFE3-1. The identification of CRFE3-1 suggests that protein degradation mechanisms are important to regulate the activity of RXam1.

Development of species-specific primers for detection of Xanthomonas campestris pv. campestris causing black rot of crucifers

Aim: Black rot of crucifers caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) is a major seed-borne disease. The present study aimed to develop a rapid diagnostic protocol for the specific and sensitive detection of this pathogen. Methodology: A specific primer set was designed based on rpf gene and optimization of PCR condition was done for specific detection of Xcc. Sensitivity of PCR for primer set was then determined by diluting the Xcc DNA and cells. Results: Specific primer set was able to amplify a specific band of 304 bp in all 11 isolates of Xcc but failed to amplify other Xanthomonas species and one each of Ralstonia solanacearum, Erwinia caratovora subsp. caratovora, Bacillus subtilis, Pseudomonas fluorescens and P. aeruginosa. The primer set was highly sensitive as it was able to detect 10 pg μl-1 bacterial DNA and up to 3x103 CFU ml-1 corresponding to 12 viable cells of Xcc which were used as template for PCR reaction Interpretation: The results suggest that developed PCR primers are highly specific and sensitive and it can be used to detect the pathogen at an early stage of infection for disease management.

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

MarR family transcription regulator HpaR and XC0449 coordinately regulate the virulence of Xanthomonas campestris pv. campestris / 生物工程学报

MarR family transcription regulators are ubiquitous among bacteria and archaea. They extensively control multiple cellular processes and elaborately regulate the expression of genes involved in virulence, stress response and antibiotics at translational level. In Xanthomonas campestris pv. campestris, insertional inactivation of MarR family transcription regulator HpaR (XC2827) resulted in significantly decrease in virulence and increase in the production of the extracellular proteases. Here, we reported that the genome of Xcc 8004 encodes nine MarR family transcription regulators. The MarR family transcription regulators, HpaR (XC2827) and XC0449, were heterologous expressed and purified. In vitro MST and Pull-down assay confirmed the physical interaction between HpaR and XC0449. Phenotypical assay determined that deletion of XC0449 resulted in substantial virulence attenuation. In vitro EMSA, in vivo qRT-PCR and GUS activity assay identified that HpaR and XC0449 coordinately act as the transcriptional activator to regulate the expression of the virulence-associated gene XC0705, and eventually control the bacterial virulence and the production of extracellular proteases.

ESTUDIO DE LA EXPRESIÓN DE GENES QUE CODIFICAN PARA PUTATIVAS PROTEÍNAS PR EN YUCA (Manihot esculenta Crantz) / Studying the Expression of Genes Coding for Putative PR Proteins in Cassava (Manihot esculenta Crantz)

RESUMEN Posterior al reconocimiento de agentes patógenos las plantas activan una serie de cascadas de señalización que culminan con la activación de factores de transcripción. Esto genera una concomitante reprogramación de la expresión génica que incluye la activación de la transcripción de los genes PR (relacionados con patogenicidad). Las proteínas PR son conocidas por poseer actividad antimicrobiana y evitan la posterior colonización del patógeno. En este estudio se empleó una aproximación bioinformática para identificar el repertorio de posibles proteínas PR en el genoma de yuca. Adicionalmente, se evaluó la expresión de nueve genes PR a lo largo del tiempo en variedades de yuca resistentes y susceptibles en respuesta a la inoculación con la bacteria Xanthomonas axonopodis pv. manihotis (Xam) mediante RT-PCR. Se encontró que varios genes PR fueron inducidos producto de la herida que se realiza durante el proceso de inoculación. Con el fin de evaluar cuantitativamente la contribución real de la infección bacteriana en la expresión de estos genes, se llevó a cabo una RT-PCR en tiempo real (QRT, Quantitative Real-Time PCR). Se encontró que en la variedad resistente el gen que codifica para MePR1 (Manes06G026900.1) presentó una inducción en su expresión a diferentes tiempos post-inoculación, lo cual no se observó en la variedad susceptible. De esta manera, este gen se constituye en un excelente marcador para evaluar la respuesta molecular de resistencia en plantas de yuca.

ABSTRACT Once pathogens are perceived by plants a signal transduction pathway is activated leading to the induction of transcription factors, which in turn reprogram the host gene expression including the transcription of PR (Pathogenesis-Related) genes. The PR proteins are well known for their antimicrobial activity and for contributing to arrest the invasion of pathogens. In this work, a bioinformatics approach was used to identify the repertoire of possible PR proteins in the cassava genome. Additionally, the expression of nine PR genes was evaluated over a time course in resistant and susceptible cassava varieties in response to inoculation with the bacterium Xanthomonas axonopodis pv. manihotis (Xam) by semiquantitative RT-PCR. It was found that several PR genes were induced as a result of the wound that is made during the inoculation process. In order to evaluate quantitatively the real contribution of the bacterial infection in the expression of the genes, a Real Time RT-PCR (qRT, quantitative Real-Time PCR) was carried out. In the resistant variety the gene coding for MePR1 (Manes06G026900) was induced at different post-inoculation times, which was not observed in the susceptible variety. Therefore, this gene constitutes an excellent marker to evaluate the molecular resistance response in cassava plants.

Reaction of yellow passion fruit to passion fruit woodiness disease and to bacterial spot / Reação de maracujazeiro azedo à virose do endurecimento dos frutos e à bacteriose

The use of resistant varieties is a promising strategy for passion fruit woodiness disease (PWD) (Cowpea aphid-borne mosaic virus - CABMV) and bacterial spot (Xanthomonas axonopodis pv. passiflorae - Xap) control in yellow passion fruit (Passiflora edulis Sims). This study aimed at evaluating the reaction of nine genotypes of yellow passion fruit to both mechanically inoculated CABMV and Xap, under protected cultivation. The experiment was conducted using a randomized block design with subdivided parcels comprised of nine treatments, four repetitions, six replications per genotype, and five evaluations. Disease incidence (% plants infected) and severity (% of total leaf area with necrotic lesions or leaf symptoms) were calculated at 7-day intervals. All genotypes evaluated were classified as moderately susceptible to PWD. MAR20#10, MAR20#41, and Rosa Intenso were classified as moderately resistant to bacterial spot whereas the remaining genotypes were classified as moderately susceptible. Genotypes Rosa Intenso, MAR20#41, MAR20#15, and MSCA stood out for presenting the lowest PWD mean severity scores as well as the greatest numbers of plants presenting resistance to PWD after five evaluations. MAR20#10, MAR20#41, and Rosa Intenso demonstrated the lowest bacterial spot mean severity scores. Among the genotypes selected, Rosa Intenso and MAR20#41 were the most promising genotypes for presenting the lowest severity scores for both PWD and bacterial spot disease.

O uso de variedades resistentes é uma estratégia promissora para o controle da virose do endurecimento dos frutos (VEF) (Cowpea aphid-borne mosaic virus - CABMV) e da bacteriose (Xanthomonas axonopodis pv. passiflorae - Xap) no maracujazeiro amarelo (Passiflora edulis Sims). Este estudo objetivou avaliar a reação de nove genótipos de maracujazeiro amarelo à CABMV e Xap, ambos inoculados mecanicamente, sob cultivo protegido. O experimento foi conduzido em delineamento de blocos casualizados com parcelas subdivididas, composto por nove tratamentos, quatro repetições, seis plantas por genótipo e cinco avaliações. A incidência (% plantas infectadas) e a severidade (% da área foliar total com lesões necróticas ou sintomas foliares) das doenças foram calculadas em intervalos de sete dias. Todos os genótipos avaliados foram classificados como moderadamente suscetíveis à VEF. MAR20#10, MAR20#41 e Rosa Intenso foram classificados como moderadamente resistente à bacteriose enquanto os demais genótipos foram classificados como moderadamente suscetíveis. Os genótipos Rosa Intenso, MAR20#41, MAR20#15 e MSCA se destacaram por apresentarem menores severidades médias da VEF bem como pelo maior número de plantas apresentando resistência à virose após as cinco avaliações. MAR20#10, MAR20#41 e Rosa Intenso demonstraram as menores severidades médias de bacteriose. Entre os genótipos selecionados, Rosa Intenso e MAR20#41 foram os mais promissores por apresentarem os menores valores de severidade para a VEF e para a bacteriose.

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

Yield and rheological properties of exopolysaccharide from a local isolate: Xanthomonas axonopodis pv. vesicatoria

Background: The aim of the present study was to evaluate gum productivity of a local strain, Xanthomonas axonopodis pv. vesicatoria, isolated from pepper plant, and its rheological behavior for the first time compared to the standard strain, Xanthomonas campestris DSM 19000 (NRRL B-1459). The influence of operational conditions (agitation rate and inoculum volume) on gum production and rheological properties of gums from the Xanthomonas strains were investigated. Results: The isolated strain of Xanthomonas showed similar xanthan yield compared to the standard strain. Furthermore, this study clearly confirmed that gum yield depended on bacterial strain, agitation rate, and inoculum size. The most suitable conditions for the gum production in an orbital shaker in terms of agitation rate and inoculum size were 180 rpm and 5%, respectively, resulting in an average production of 10.96 and 11.19 g/L for X. axonopodis pv.vesicatoria and X. campestris DSM 19000, respectively. Regarding the rheological properties, Ostwald-de-Waele and power law models were used to describe flow and oscillatory behavior of the gum solutions, respectively. Consistency of the novel gum solution remarkably was much higher than the commercial xanthan gum solution. Flow and oscillatory behavior and their temperature ramps showed that weak gel-like structure could be obtained with less gum concentrations when the novel gum was used. Conclusion: Therefore, yield and technological properties of the aqueous solutions of the exopolysaccharide synthesized by X. axonopodis pv. vesicatoria were observed to be more suitable for industrial production.