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.

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.

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.

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.

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.

Identificación de QTL de resistencia a la bacteriosis vascular en yuca bajo condiciones naturales de infección / Qtl identification for cassava bacterial blight resistance under natural infection conditions

Cassava, Manihot esculenta Crantz, represents the main food source for more than one billion people. Cassava's production is affected by several diseases, one of the most serious is cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam). A quantitative trait loci (QTL) analysis for CBB resistance was performed under natural infection conditions, using a mapping population of 99 full-sibs genotypes highly segregant and a SNP-based high dense genetic map. The phenotypic evaluation was carried out in Puerto López, Meta, Colombia, during the rainy season in 2015. Both resistant and susceptible transgressive segregants were detected in the mapping population. Through a non-parametric interval mapping analysis, two QTL were detected, explaining 10.9 and 12.6 % of phenotypic variance of resistance to field CBB. After a bioinformatics exploration four genes were identified in the QTL intervals. This work represents a contribution to the elucidation of the molecular bases of quantitative cassava resistance to Xam.

La yuca, Manihot esculenta Crantz, representa la principal fuente de alimento para cerca de 1000 millones de personas. La producción de yuca se ve afectada por diversas enfermedades, una de las más serias es la bacteriosis vascular (CBB) causada por Xanthomonas axonopodis pv. manihotis (Xam). En este estudio se realizó un análisis de loci de rasgos cuantitativos (QTL) para la resistencia a CBB en condiciones naturales de infección, usando una población de mapeo constituida por 99 genotipos de hermanos completos segregantes y un mapa genético altamente denso basado en SNPs. La evaluación fenotípica se llevó a cabo en Puerto López (Meta), Colombia, durante la época de lluvias durante el segundo semestre de 2015. En la población de mapeo fueron detectados individuos con una segregación transgresiva tanto resistentes como susceptibles. A través de un análisis no paramétrico de intervalo simple, se detectaron dos QTL que explican el 10,9 y el 12,6 % de la varianza fenotípica de la resistencia en campo a CBB. Mediante análisis bioinformáticos se identificaron cuatro genes candidatos presentes en los intervalos de los QTL. Este trabajo representa un esfuerzo por dilucidar los mecanismos moleculares implicados en la resistencia de yuca a CBB.

Xanthomonas fuscans subsp. fuscans causing wilt symptoms in bean plants (Phaseolus vulgaris) in Brazil / Xanthomonas fuscans subsp. fuscans causando sintomas de murcha em feijoeiro (Phaseolus vulgaris) no Brasil

Common and fuscous blights of bean are diseases widely distributed in the world. The most commonly observed symptoms are spots on leaves, stems, pods and seeds. In December 2009, bean plants cv. Uirapuru showing symptoms of wilt similar to those induced by Curtobacterium flaccumfaciens pv. flaccumfaciens were observed in a commercial crop located in the county of Itararé, State of São Paulo, Brazil. The plants were at the last cycle stage with mature pods and these symptoms were noted in the majority of the growing area. Optical microscopic observations of discolored vascular tissue from diseased stems revealed the presence of bacterial masses oozed from infected tissue, indicating that the disease was caused by bacterial pathogen. Isolations on nutrient agar showed circular, convex, yellow colonies with smooth edges. The causal bacterium was Gramnegative and produced a dark brown pigment in culture medium. Biochemical, cultural and physiological tests confirmed its identity as Xanthomonas fuscans subsp. fuscans(syn. Xanthomonas campestris pv. phaseoli"var. fuscans"). The pathogenicity of the isolates was confirmed by artificial inoculations. Systemic infection has been reported in the literature but these kinds of symptoms are not currently observed in Brazilian fields. Bacterial strains were deposited on the Phytobacteria Culture Collection of Instituto Biológico (IBSBF - www.biologico.sp.gov.br/bacterias/php) under accession numbers 2813 and 3028.(AU)

O crestamento bacteriano comum do feijoeiro é uma doença amplamente distribuída no mundo e os sintomas comumente observados são manchas nas folhas, hastes, vagens e sementes. Em dezembro de 2009, plantas de feijoeiro cv. Uirapuru com sintomas de murcha similares aos produzidos por Curtobacterium flaccumfaciens pv. flaccumfaciens foram observadas em campos comerciais localizados no município de Itararé, estado de São Paulo, Brasil. As plantas encontravam-se no final do ciclo, com as vagens já formadas. Os sintomas foram notados em quase a totalidade da área cultivada. Observações ao microscópio óptico de fragmentos de tecido do sistema vascular das hastes de plantas doentes evidenciaram intenso fluxo bacteriano, confirmando tratar-se de doença bacteriana. Isolamentos realizados em meio nutriente ágar produziram colônias de coloração amarelada, brilhantes, convexas, lisas. A bactéria agente causal era Gram-negativa e produtora de pigmento marrom escuro em meio de cultura. Testes bioquímicos, culturais e fisiológicos confirmaram sua identidade como Xanthomonas fuscans subsp. fuscans (sin. Xanthomonas campestris pv. phaseoli "var. fuscans"). A patogenicidade dos isolados foi confirmada por inoculações artificiais em mudas de feijão cv. Carioca e os reisolamentos efetuados resultaram em colônias semelhantes às originais. Embora descrita na literatura, a infecção sistêmica não é usualmente observada nos plantios de feijoeiro em nosso país. Linhagens bacterianas encontram-se depositadas na Coleção de Culturas do Instituto Biológico (IBSBF) sob os números 2813 e 3028.(AU)

Genótipos de maracujazeiro-azedo com resistência à bacteriose / Genotypes of passionfruit to resistance to the bacteriose

Este trabalho teve como objetivo avaliar e selecionar genótipos de maracujazeiro-azedo com resistência à bactéria Xanthomonas axonopodis pv. passiflorae. Para a inoculação da bactéria, nas folhas das plantas, foi empregado o método da agulha utilizando-se o delineamento de blocos casualizados, com 4 repetições e 12 plantas por parcela, em esquema de parcela subdividida, com quatro épocas de avaliação na parcela e 18 genótipos na subparcela. Foi avaliada a incidência e a severidade da bacteriose, com base em escala de notas variando de 0 a 4, onde foram classificadas como Resistentes, Medianamente Resistentes, Medianamente Suscetíveis, Suscetíveis e Altamente Suscetíveis. Os genótipos apresentaram variabilidade com relação à resistência, sendo que os genótipos Maracujá Moranga, RC-0-3, Vermelhinho e PES-7 foram selecionados, pois apresentaram mais de 30% de plantas medianamente resistentes na última avaliação. O genótipo MSCA foi considerado resistente com base na severidade. As plantas individuais dos diferentes genótipos que permaneceram resistentes até a última avaliação foram selecionadas e deverão ser novamente avaliadas para a confirmação da resistência à bactéria Xanthomonas axonopodis pv. passiflorae, incluindo diferentes isolados desta bactéria.

This work had as objective to evaluate and to select the reaction of passionfruit genotypes to Xanthomonas axonopodis pv. passiflorae. For the inoculation of the bacterium had been used of the needle. Twelve plants of each of 18 genotypes were evaluated in a randomized block experiment with 4 replicates and four different times of evaluation. It was evaluated the incidence and the severity of bacteriose, on the basis of a note scale of 0 the 4 which were classified as Resistant, Medium Resistant, Medium Susceptible, Susceptible and Highly Susceptible. The genotypes had presented variability with regard to the resistance, being that the genotypes Maracujá Moranga, RC-0-3, Vermelhinho and PES-7 had been selected, since they had more than presented 30% of medium resistant plants in the last evaluation. Only one genotype was considered resistant, on the basis of severity. The individual plants of the different genotypes that had remained resistant until the last evaluation will have to be selected and evaluated for the confirmation of the Xanthomonas axonopodis pv. passiflorae.

Elaboração e validação de escalas diagramáticas para avaliação da severidade da mancha-bacteriana do feijão - caupi em cultivares com trifólios morfologicamente distintos / Elaboration and validation of a diagrammatic scale to assess bacterial blight severity on cowpea cultivars with morphologically distinct leaflets

Duas escalas diagramáticas foram desenvolvidas para quantificar a severidade da mancha-bacteriana do feijão-caupi, causada pela bactéria Xanthomonas axonopodis pv. vignicola, em cultivares de feijão-caupi de trifólios com folíolos ovais e lanceolados. Trifólios com diferentes níveis de severidade foram coletados para determinação do limite mínimo e máximo de severidade, sendo níveis intermediários determinados de acordo com a "Lei do Estímulo de Weber-Fechner". Os níveis propostos foram: 0,05; 0,25; 1,00; 3,40; 9,70; e 17,70% para trifólios com folíolos ovais e 0,10; 0,35; 1,10; 3,50; 9,30; e 19,10% para trifólios com folíolos lanceolados. As escalas foram validadas por meio do cálculo da sua acurácia e precisão. Para tanto, realizou-se análises de regressão linear da severidade real com a estimada com e sem o uso das escalas propostas. As estimativas foram feitas por 12 avaliadores sem experiência em quantificar essa doença em 50 trifólios com diferentes níveis de severidade da mancha-bacteriana. Para isso, avaliou-se a acurácia e a precisão dos avaliadores. Com a utilização das escalas diagramáticas, obtiveram-se níveis satisfatórios de acurácia e precisão. Assim, as escalas propostas tornam-se uma ferramenta fácil e de rápida utilização, que podem ser adotadas para auxiliar na avaliação da severidade da mancha-bacteriana do feijão-caupi, em estudos epidemiológicos e seleção de cultivares resistentes a doença.

Two diagrammatic scales were developed to assess the severity of bacterial blight of cowpea, caused by Xanthomonas axonopodis pv. vignicola in cowpea cultivars with oval and lanceolate leaflets, considering the limits for minimum and maximum severity of the disease, as well as intermediate levels. The levels were determined in accordance with the "Weber-Fechner's Stimulus Responses Law", representing: 0.05; 0.25; 1.00; 3.40; 9.70; and 17.70% for diagrammatic scale with oval leaflets and 0.10; 0.35; 1.10; 3, 50; 9.30; and 19.10% for lanceolate leaflets. The scales were validated by means of their accuracy and precision. To calculate these factors, linear regression analysis of actual disease severity and the estimated ones with and without the use of the scales. Twelve non-experienced evaluators estimated 50 leaflets with different levels of severity bacterial blight. For this, the accuracy and precision of the evaluators was assessed. With the use of diagrammatic scales, there were satisfactory levels of accuracy and precision. Thus, the diagrammatic scales proposed were considered appropriate to assess the severity of bacterial blight of cowpea and can be used in epidemiological studies, to evaluate the reactions of genotypes to the disease.

In planta detection of Xanthomonas axonopodis pv. commiphorae using fyuA and rpoD genes.

Guggal is tapped for extraction of medicinally important oleo–gum–resin (guggul) by inoculating the stem bark with natural gum suspension containing pathogenic bacterium Xanthomonas axonopodis pv. commiphorae (Xac). The tree dies in the process. In absence of any specific medium for isolation of Xac, it is difficult to assess spread of the pathogen within the plant. A PCR based molecular detection technique using fyuA and rpoD gene specific primers is described here. The primers amplified products only from Xac and not from host tissues or common saprophytes. The method was sensitive enough to produce positive signals for up to 4.4 bacterial cells or 2 pg target DNA per reaction mixture. However, PCR inhibitors present in plant tissues drastically reduced the limit of detection. A simple overnight incubation of surface sterilised plant tissues in nutrient medium was introduced to increase pathogen titre and to overcome this problem. This technique was successfully used to measure spread of Xac in plant tissues away from the site of inoculation. The pathogen showed preference for acropetal movement and did not spread to 7–8 cm below the site of inoculation till 15 days after inoculation. This suggests possibility to manage the disease through plant surgery.

Reação de genótipos de feijão ao crestamento bacteriano comum, avaliado por dois métodos de inoculação / Reaction of bean genotypes to the common bacterial blight, evaluated by two inoculation methods

Conhecer a resistência de genótipos de feijão à fitopatógenos é importante para uso em programas de melhoramento genético. Duas técnicas de inoculação bacteriana foram testadas para avaliar a sensibilidade de genótipos de feijão ao crestamento bacteriano comum, causado por Xanthomonas axonopodis pv. phaseoli. Foram inoculados folíolos de 56 genótipos de feijão, utilizando-se das técnicas de agulhas múltiplas e incisão com tesoura, utilizando o isolado XAP 15497. Foi possível classificar 21 genótipos resistentes, 13 moderadamente resistentes, 9 moderadamente suscetíveis e 13 suscetíveis pelo método de inoculação com agulhas múltiplas. No método de inoculação com tesoura, foi possível classificar 24 genótipos como resistentes, 21 como moderadamente resistentes, 7 como moderadamente suscetíveis e 4 suscetíveis. Os genótipos IPR Chopim, IAPAR 81, IAPAR 16, BRS Campeiro, BRS Radiante, IAPAR 80, UTF 6, SM 9906, PI 2072620, LP 01-51, PB 4, Uirapuru, IAPAR 20, Zonin, IAPAR 31, 2 V, CNPF 7762, BRS Talismã, Pérola, Lon Rosinha e UTF 9 tiveram comportamento semelhante diante dos dois métodos de inoculação. Verificou-se que os dois métodos de inoculação foram eficientes para a determinação das reações de suscetibilidade e resistência dos genótipos. Porém, a técnica de inoculação com agulhas múltiplas demonstrou maior praticidade e precisão para a avaliação da área foliar inoculada.

The knowledge of resistance of bean genotypes to pathogens is important to genetic breeding programs. Two methods of bacterial inoculation were tested to evaluate the sensitivity of bean genotypes to common bacterial blight, caused by Xanthomonas axonopodis pv. phaseoli. Leaves of 56 bean genotypes were inoculated by the method of multiple needles and the method of incision with scissors, using the isolated XAP 15497. We classified 21 genotypes as resistant, 13 as moderately resistant, 9 as moderately susceptible, and 13 as susceptible by the method of inoculation with multiple needles. With the method of inoculation with scissors it was possible to classify 24 genotypes as resistant, 21 as moderately resistant, 7 as moderately susceptible, and 4 as susceptible. The genotypes IPR Chopim, IAPAR 81, IAPAR 16, BRS Campeiro, BRS Radiante, IAPAR 80, UTF 6, SM 9906, PI 2072620, LP 01-51, PB 4, Uirapuru, IAPAR 20, Zonin, IAPAR 31, 2 V, CNPF 7762, BRS Talismã, Pérola, Lon Rosinha, and UTF 9 had similar behavior in the two methods of inoculation. Both methods of inoculation were efficient in determining the reactions of susceptibility and resistance of the genotypes but the method of inoculation with multiple needles was more precise and practical in the evaluation of the inoculated portion of the leaf.

Produção de frutos e incidência de cancro cítrico em laranjeiras "Monte Parnaso" enxertadas sobre sete porta-enxertos / Fruit production and incidence of citrus canker lesions on “Monte Parnaso” navel orange budded on seven rootstocks

Porta-enxertos de citros podem exercer influência sobre a produção de frutos e suscetibilidade das plantas ao cancro cítrico. Este trabalho objetivou selecionar porta-enxertos que induzam elevado índice de produtividade e menor suscetibilidade das copas de laranjeiras-de-umbigo "Monte Parnaso"(Citrus sinensis) à bactéria Xanthomonas axonopodis pv. citri, causadora do cancro cítrico (CC). O experimento foi desenvolvido em um pomar experimental com 10 anos de idade, plantado em espaçamento de 2,5 x 6,0m, no município de Butiá, no Estado do Rio Grande do Sul. O delineamento experimental foi em blocos ao acaso, com sete tratamentos e quatro repetições, usando duas plantas por parcela. Os tratamentos constaram dos seguintes porta-enxertos: limoeiros "Cravo" (C. limonia) e "Volkameriano" (C. volkameriana.), trifoliata "Flying Dragon" (Poncirus trifoliata.), laranjeira "Caipira" (C. sinensis), citrangeiro "Troyer" (C. sinensis x P. trifoliata), tangerineira "Sunki" (C. sunki.) e citrumeleiro "Swingle" (C. paradisi x P. trifoliata). Os parâmetros avaliados foram: incidência de CC em folhas e frutos em toda a planta e em ramos previamente selecionados, em diversas épocas. Verificou-se que as plantas enxertadas sobre citrumeleiro "Swingle" apresentaram o maior índice de produtividade e, à semelhança do "Flying Dragon", a menor incidência de CC, tanto nas folhas como nos frutos. Além disso, verificou-se também que as plantas enxertadas sobre os limoeiros "Cravo" e "Volkameriana", apesar de produzirem as mais elevadas cargas de frutos, foram as mais suscetíveis ao CC.

Citrus rootstocks can exert some influences on fruit production and susceptibility of the plants to citrus canker (CC). This research was performed aiming to select rootstocks which induce higher productivity index and lower susceptibility of 'Monte Parnaso' navel orange (Citrus sinensis) trees to the bacteria Xanthomonas axonopodis pv. citri. The research was carried out in an experimental orchard, with ten-years-old trees, planted at spacement of 2.5m between trees and 6.0m between rows, located in Butiá city, in the Central Depression of Rio Grande do Sul state. It was used an randomized blocks experimental design, with 7 treatments and 4 replications. The treatments consisted of the following rootstoks: Rangpur lime (C. limonia.), 'Volkameriana' lemon (C. volkameriana.), 'Flying Dragon' trifoliata orange (Poncirus trifoliata), 'Caipira' orange (C. sinensis), 'troyer' citrange (C. sinensis x P. trifoliata), 'Sunki' tangerine (C. sunki.) and 'Swingle' citrumelo (C. paradisi x P. trifoliata). The parameters evaluated were: number of fruits and leaves with citrus canker lesions per plant and at two previously selected and marked branches per plant, at different times . The results revealed that the 'Swingle' citrumelo and 'Flying dragon' rootstocks induced the highest productivity index and, the lowest incidence of citrus canker disease on leaves and fruits. 'Rangpur' lime and 'Volkameriana' lime rootstocks, promoted a heavy crop load, however, showed higher susceptibility to citrus canker disease.

Rizobactérias no controle da mancha angular do algodoeiro / Rhizobacteria to control cotton bacterial blight

Avaliou-se o potencial de rizobactérias na indução de resistência do algodoeiro à Xanthomonas axonopodis pv. malvacearum. Após o isolamento das rizobactérias, foram selecionados os isolados capazes de reduzir os sintomas da mancha angular bacteriana em casa de vegetação, os quais foram aplicados espacialmente separados do patógeno desafiador. Os melhores isolados foram testados quanto à capacidade de reduzir os sintomas da ramulose e da murcha de Verticillium e de inibir diretamente os patógenos in vitro. Do total de 123 isolados de rizobactérias foram selecionados cinco, L2-1 (Bacillus cereus), MT5-6 (Bacillus cereus), L2-2 (Achromobacter xylosoxidans), MT5-5 (Bacillus cereus) e MT5-11 (Brevibacterium sp.), os quais apresentaram controle da mancha angular acima de 40 por cento, em relação à testemunha. Nenhum isolado reduziu a severidade da ramulose e da murcha de Verticillium em relação à testemunha, nem apresentou efeito inibitório direto in vitro a X. axonopodis pv. malvacearum e Colletotrichum gossypii var. cephalosporioides. Para V. dahliae, apenas o isolado L2-1 apresentou efeito inibitório.

The potential of rhizobacteria was evaluated for resistance induction against Xanthomonas axonopodis pv. malvacearum. After isolation, the rhizobacteria were screened for the reduction of angular leaf spot severity under greenhouse conditions. They were spatially separated from the challenging pathogen. The best isolates were tested for the capacity to reduce ramulose and Verticillium wilt severity and directly inhibit pathogens in vitro. From a total of 123 rhizobacterial isolates, five were selected, L2-1 (Bacillus cereus), MT5-6 (Bacillus cereus), L2-2 (Achromobacter xylosoxidans), MT5-5 (Bacillus cereus) and MT5-11 (Brevibacterium sp.), which showed angular leaf spot control above 40 percent as compared to the control. The tested isolates neither reduced the severity of ramulose and verticillium wilt compared to the control nor showed in vitro direct inhibition to X. axonopodis pv. malvacearum and Colletotrichum gossypii var. cephalosporioides. For V. dahliae, only isolate L2-1 showed direct inhibition.

Reação de germoplasma de mandioca a Xanthomonas axonopodis pv. manihots / Reaction of cassava’s germoplasm to Xanthomonas axonopodis pv. manihots

A mandioca apresenta-se como importante fonte de carboidratos, principalmente nos trópicos. A bacteriose causada por Xanthomonas axonopodis p.v. manihots é a doença mais importante desta cultura e seus danos podem chegar a 30% ou mais na produção. Este trabalho objetivou avaliar, em condições de casa de vegetação, a reação de germoplasma de mandioca mansa e de brava à dois isolados de Xanthomonas axonopodis p.v. manihots. Os ensaios foram desenvolvidos no Instituto de Ciências Agrárias da Universidade Federal de Uberlândia. As plantas foram inoculadas pelo corte de três folíolos centrais em três folhas novas completamente abertas, seguindo pela inserção de um palito à altura da axila da folha mais velha, utilizando-se uma suspensão bacteriana contendo 2x109 u.f.c. mL-1 A inoculação ocorreu aos 42 dias de plantio e a avaliação aos 41 dias de inoculação. Os critérios de avaliação foram notas de Sintomas visuais na parte aérea, Porcentagens de desfolha e de Infecção sistêmica do caule. Os resultados mostraram que os critérios de avaliação utilizados foram eficientes no estudo da virulência dos isolados, sendo o isolado obtido na região de Uberlândia mais virulento para mandioca mansa e o de Lavras para mandioca brava, caracterizando a necessidade de utilização de isolados provenientes da região onde o germoplasma será cultivado. Considerando-se a análise da reação da resistência de germoplasma, os critérios porcentagem de infecção sistêmica e de desfolha mostraram-se bastante efetivos. Destacaram-se nesse trabalho as variedades Vassoura, Amarela, Vermelha e Castelinho e o clone CPAC88-11.

The cassava is presented as an important stach source, mainly in the tropics. The bacteriosis diseasecaused by Xanthomonas axonopodis p.v. manihots is the most important disease of this culture and itsdamage can achieve 30% of the production, or even more. This work objectified to evaluate, in greenhouse condition the reaction of "mandioca mansa" and the "mandioca brava" cassava's germoplasma tothe two isolates of Xanthomonas axonopodis p.v. manihots. Trials were developed at the Instituto deCiências Agrárias da Universidade Federal de Uberlândia. The plants had been inoculated by seasor'scuttings of three central leaflets in three completely opened new leaves, following by insertion of a littlewood stick at the oldest leaf's axel, using a bacterial suspension at 2x109u.f.c. mL-1. The inoculation happened at the 42nd day after planting and the evaluation at the 41st day after inoculation. Theevaluation criteria were: notes of visual symptoms in the aerial part, percentages defoliation and systemicinfection of the stalk. The results showed the efficiency of the evaluation criteria applied at this work forthe isolates virulence study. The Uberlândia isolate was more virulent to "mandioca mansa" cassavacultivars and the Lavras isolate was more virulent to "mandioca brava" cassava cultivars. That indicatesthe need of using isolates from the region where the germoplasm will be cultivated. Considering thegermoplasm resistance reaction analysis, both the sistemic infection precentage and the defoliationcriteria presented as very effective. Oustanding behavior was observed for the Vassoura, Amarela,Vermelha and Castelinho cultivars and for the CPAC88-11 clone