Isolation and identification of a new phytopathogen causing root rot of Rehmannia glutinosa / 中国中药杂志

Root rot was occurred widely in the production area of Rehmannia glutinosa, and which result in serious influence on the yield and quality of R. glutinosa. In the present work, a new phytopathogen was isolated from roots with root rot symptom in the production area of R. glutinosa. The colony of the pathogen growing on PDA medium was gray-black, the structure of hyphae was compact, the aerial hyphae was less developed, and the back of the colony was black. The hyphae of the pathogen were uneven in size, about 2 to 3 μm in diameter and twined with each other, the conidia of the pathogen were small, nearly round and about 1 μm in diameter. The healthy roots of R. glutinosa were inoculated with the pathogen in vitro, black-brown rot was observed at the inoculate sites after a few days' incubation. The rhizosphere soil of healthy R. glutinosa seedlings were inoculated in vivo, the leaves were wilted and the roots were black-brown rotted after several days' normal culture, the symptoms were consistent with those observed in the field. The genomic DNA of the pathogen was amplified by fungus rDNA-ITS universal primer ITS1/ITS4 and homologous analyzed, the pathogen was in a branch with Heterophoma sp., Phoma sp., P. novae-verbascicola and P. herbarum with the nuclear acid homology of 99.21% to 99.43%. The pathogen shown 97.00% to 98.02% nuclear acid homology with H. verbascicola, H. novae-verbascicola, H. poolensis, P. herbarum, H. sylvatica, H. verbascicola and H. verbasci-densiflori when amplified by the tub2 gene special primer Btub2 fd/Btub4 rd, and H. novae-verbascicola was the highest. The pathogen was in a branch with H. novae-verbascicola when amplified by the lsu gene special primer LR0 R/LR7. Based on the morphological characteristics, nucleotide sequence analysis and Koch's test results, the isolated pathogen causing root rot of R. glutinosa was identified as H. novae-verbascicola. This study is of great significance for the further theoretical research on root rot of R. glutinosa and root rot control in field.

Antifungal activity of homeopathic medicines against the white mold causing agent Sclerotinia sclerotiorum

White mold disease, caused by fungus Sclerotinia sclerotiorum (Lib.) de Bary., is a disease hard to control due to the high amount of sclerotia produced, which guarantees its survival in the soil for years leading to significant yield losses. Alternative techniques to control the pathogen have been researched, including homeopathy. The present work aimed to evaluate the in vitro antifungal effect of homeopathic medicines on S. sclerotiorum mycelial growth. Homeopathic medicines Sulphur, fungal sclerotium Nosode and Calcarea carbonica, in 30CH, 200CH and 1000CH dynamizations were tested. Assays were carried out in a completely randomized design, with four repetitions. Experiments were performed through the addition of homeopathic medicines on the surface of plates containing culture medium, followed by insertion of a disc containing fungus mycelia and incubation. Control treatment received no homeopathic medicine. The mycelial progression was monitored by seven halo diameter measurements during experiment period. All homeopathic medicines tested and their dynamizations were able to inhibit partially the development of the fungus. Calcarea carbonica at the dynamization of 1000 CH showed the best inhibitory effect on S. sclerotiorum, which under its effect produced a mycelial halo 40% smaller than the control treatment.

IDENTIFICACIÓN MOLECULAR Y REGISTRO DE Aphelenchoides spp. EN CULTIVOS COMERCIALES DE Hydrangea EN ANTIOQUIA, COLOMBIA / MOLECULAR IDENTIFICATION AND REPORT OF Aphelenchoides spp. IN COMMERCIAL CROPS OF Hydrangea IN ANTIOQUIA, COLOMBIA

RESUMEN El cultivo comercial de hortensias para flor de exportación ocupa un renglón importante en el sector económico del oriente antioqueño, por ser fuente de empleo y de desarrollo en la zona. La hortensia (Hydrangea macrophylla) es afectada por numerosos organismos fitopatógenos, entre ellos, nematodos del género Aphelenchoides, los que ocasionan, en el follaje, lesiones necróticas angulares, malformación de flor, enanismo y un daño indirecto en la tasa fotosintética, demeritando los parámetros de calidad para exportación. El objetivo de este estudio fue identificar, molecularmente, las especies del nematodo Aphelenchoides asociadas al cultivo de hortensias de color, en los municipios de Medellín (Santa Elena), La Ceja y Rionegro, siendo este el primer reporte para Colombia, de las especies de este género. Para la ejecución del estudio, se realizaron 10 muestreos en cultivos comerciales, distribuidos entre los tres municipios mencionados. Los nematodos extraídos, se sometieron a pruebas basadas en el análisis de ADN, haciendo uso del marcador ribosomal 18S. Los análisis filogenéticos practicados mostraron la presencia de la especie Aphelenchoides ritzemabosi en cultivos de hortensias, del corregimiento de Santa Elena y, de A. fragarie, en los municipios de La Ceja y Rionegro.

ABSTRACT Hydrangea macrophylla commercial crops have an important economic significance for the eastern region of the department of Antioquia, Colombia, due to its impact on employment and local development. This flowering plant can be attacked by several phytopathogens among them nematodes of the genus Aphelenchoides; which can produce necrotic lesions on the plant leaves as well decreased photosynthetic rate, dwarfism, and flower malformation. Additionally, this pathogen is considered a regulated pest and limits the international commercialization of the flower. The aim of this study was to molecularly identify the Aphelenchoides species associated with infected H. macrophylla plants in crops the municipalities of Medellín, La Ceja, and Rionegro. This is the first report of the species of this phytopathogen for Colombia. The sampling activities were performed in ten commercial crops locate at the three municipalities. The isolated nematodes were subject to DNA-based tests were the 18S rDNA gene was amplified, sequenced and analyzed using phylogenetic methods. The obtained results showed the infection of Aphelenchoides ritzemabosi within the Santa Elena (Medellin) area in crops and A. fragarie within the municipalities of La Ceja, and Rionegro.

Caracterização bioquímica e funcional da proteína XadA3 de Xylella fastidiosa / Biochemistry and functional characterization of Xylella fastidiosa XadA3 adhesin

Gram-negativas e é utilizado por diversos patógenos para colonizar seus hospedeiros, sendo o primeiro passo do processo de desenvolvimento do biolfilme. Uma variedade de apêndices celulares e proteínas está envolvida na adesão bacteriana, tais como pili, fimbrias, adesinas fimbriais e afimbriais. O fitopatógeno Xylella fastidiosa, agente causal de importantes doenças como a doença de Pierce de videiras, a clorose variegada dos citros e a síndrome do rápido declínio de oliveiras, possui em sua superfície várias dessas estruturas que são potencialmente responsáveis pela colonização eficiente de insetos-vetores e plantas hospedeiras. Entre as adesinas afimbriais codificadas no genoma dessa bactéria, três XadA (XadA1, Hsf/XadA2 e XadA3) são classificadas como autotransportadores triméricos. Dados da literatura sugerem que XadA1 e XadA2 são importantes para a formação do biofilme, porém a função de XadA3 ainda não havia sido investigada. Nesse trabalho, tivemos como objetivo caracterizar bioquímica e funcionalmente a proteína XadA3 e obter informações adicionais sobre o papel desempenhado por XadA1 e XadA2 na adesão e virulência de X. fastidiosa. Utilizando imunodetecção com um anticorpo policlonal anti-XadA3 por nós obtido, demonstramos que essa proteína localiza-se na superfície bacteriana e medeia a adesão intercelular. A caracterização dos fenótipos de mutantes de deleção de cada um dos genes das adesinas XadA revelou que o mutante ΔxadA3 tem reduzida capacidade de agregação celular e formação de biofilme quando comparado tanto aos mutantes ΔxadA1 e ΔxadA2 como à cepa selvagem Temecula. A deleção dos genes xadA afeta marginalmente o perfil de expressão gênica global avaliado através de RNAseq das cepas mutantes comparativamente à cepa selvagem, porém destaca-se, nas cepas mutantes, o aumento nos níveis dos transcritos de lipases/esterases. Já foi descrito que essas enzimas parecem atuar na degradação do tecido vegetal associada aos sintomas da doença de Pierce de videiras. A deleção de xadA3 resulta em um fenótipo de hipervirulência em videiras, mas também de deficiência de transmissão pelo inseto-vetor. O conjunto dos resultados obtidos nesse trabalho evidenciam o importante papel desempenhado pelas adesinas XadAs, particularmente XadA3, na adesão intercelular, no desenvolvimento do biofilme e na virulência de X. fastidiosa

Adhesion is a widely conserved mechanism of virulence among Gram-negative bacteria that is used by several pathogens to colonize their hosts, being the first step in biolfilm development. A variety of appendages and proteins are involved in bacterial adhesion, such as pili, fimbriae, fimbrial and afimbrials adhesins. The phytopathogen Xylella fastidiosa, causal agent of important diseases such as Pierce's disease of grapevines, citrus variegated chlorosis and olive quick decline syndrome, harbours on its surface several of these structures that are potentially responsible for efficient colonization of insect vectors and plant hosts. Among the afimbrial adhesins encoded in the genome of this bacterium, three XadAs (XadA1, Hsf/XadA2 and XadA3) are classified as trimeric autotransporters. Data from the literature suggest that XadA1 and XadA2 are important for biofilm formation, but XadA3 function has not been yet investigated. In this work, we aimed to biochemically and functionally characterize the XadA3 protein and gather additional information about the role played by XadA1 and XadA2 in X. fastidiosa adhesion and virulence. Using immunodetection with a polyclonal anti-XadA3 antibody, we have demonstrated that this protein localizes to the bacterial surface and mediates intercellular adhesion. Phenotypic characterization of the deletion mutants of XadA adhesins encoded genes revealed that the ΔxadA3 mutant has reduced cell aggregation capacity and biofilm formation when compared to both ΔxadA1 and ΔxadA2 mutants as well as to Temecula wild type strain. Deletion of the xadA genes marginally affects the global gene expression profile assessed by RNA-seq of the mutant strains compared to the wild-type strain, eventhough an increase in lipase/esterase transcripts levels was observed in the mutant strains. It has been reported that these enzymes appear to participate in the degradation of plant tissue that is associated with symptoms of Pierce's disease of grapevines. The deletion of xadA3 results in a phenotype of hypervirulence in grapevines but also of deficiency in insect-vector transmission. The results obtained in this work evidenced the important role played by XadAs adhesins, particularly XadA3, in X. fastidiosa intercellular adhesion, biofilm development and virulence

Caracterização do secretoma de cepas de Xylella fastidiosa / Characterization of the secretome Xylella fastidiosa strains

As doenças causadas pelo fitopatógeno Xylella fastidiosa, uma bactéria Gram-negativa, devem-se aos seus múltiplos fatores de virulência, tais como formação de biofilme, secreção de enzimas de degradação da parede celular do xilema (CWDE), expressão de proteínas de adesão e produção de vesículas de membrana externa (OMVs). Esses fatores de virulência são controlados por uma via de sinalização mediada por DSF (fatores de sinalização difusíveis de natureza lipídica) e relacionada com percepção de quórum. Nesse trabalho, tivemos como objetivo ampliar a caracterização do secretoma de cepas selvagens e mutantes de X. fastidiosa para evidenciar proteínas e metabólitos potencialmente associados à adaptação ao hospedeiro, virulência e patogenicidade. Desenvolvemos, paralelamente, três estudos empregando como abordagens metodológicas a proteômica, a metabolômica e a transcritômica. No primeiro estudo, comparamos o secretoma (exoproteoma) da cepa Temecula1 selvagem (WT) e do mutante no gene da sintase de DSF (ΔrpfF), o qual exibe fenótipo de hipervirulência em videiras. A este estudo associamos a comparação dos transcritomas dessas cepas. Os resultados mostraram que, mesmo no cultivo in vitro, X. fastidiosa expressa e secreta fatores de virulência previamente conhecidos (lipases-esterases e proteases), além de toxinas (microcinas) que, supostamente, teriam papel de controlar bactérias competidoras pelo mesmo nicho. No segundo estudo caracterizamos a composição de OMVs secretadas no cultivo in vitro por X. fastidiosa Fb7 e 9a5c (cepas isoladas de laranjeiras) e Temecula1 (cepa isolada de videira). Demonstramos que Fb7 produz até 57% mais OMVs que 9a5c e Temecula1 e identificamos um total de 202 proteínas distintas nas OMVs produzidas pelas 3 cepas, ampliando consideravelmente o número de proteínas secretadas por meio de OMVs descrito, até então, para X. fastidiosa. Entre as proteínas enriquecidas, citamos adesinas afimbriais, porinas, lipoproteínas, hidrolases (lipases/esterases, proteases e peptidases) e uma pectina-liase putativa. Destacamos a detecção da enzima L-ascorbato oxidase nas OMVs e sugerimos que esta enzima poderia atuar na depleção do ascorbato produzido pelo hospedeiro vegetal. Além disso, demonstramos, pela primeira vez, que OMVs de X. fastidiosa transportam ácidos graxos da família DSF, sugerindo um papel adicional para OMVs nesse fitopatógeno. Finalmente, no terceiro estudo verificamos alterações relevantes no perfil de metabólitos secretados por X. fastidiosa em resposta a sua interação com metabólitos secretados por Burkholderia phytofirmans, proposta como uma cepa para o biocontrole da doença de Pierce de videiras. Confirmamos que o sobrenadante de B. phytofirmans possui um composto de natureza apolar que induz a formação de biofilme em X. fastidiosa, contudo ainda não foi possível decifrar a natureza química deste composto

The diseases caused by the phytopathogen Xylella fastidiosa, a Gram-negative bacterium, are due to multiple virulence factors, such as biofilm formation, secretion of xylem cell wall degradation enzymes (CWDE), expression of adhesion proteins and production of outer membrane vesicles (OMVs). These virulence factors are controlled by a DSF (diffusible signaling factors of a lipidic nature) mediating signaling pathway and related to quorum sensing perception. In this work, we aimed to extend the characterization of the secretoma of wild type and mutants strains of X. fastidiosa to uncover proteins and metabolites potentially associated to host adaptation, virulence and pathogenicity. We developed three studies in parallel using proteomics, metabolomics and transcriptomics as methodological approaches. In the first study, we compared the secretome (exoproteome) of the wild type strain Temecula1 (WT) and of DSF synthase mutant (ΔrpfF) which exhibits hypervirulence phenotype in grapevines. We also compared the transcriptomes of these strains. Our results showed that, even in in vitro culture, X. fastidiosa expresses and secretes previously known virulence factors (lipasesesterases and proteases), as well as toxins (microcins) that might play a role in controlling competing bacteria in the same niche. In the second study, we characterized the composition of OMVs secreted by in vitro cultures of X. fastidiosa Fb7 and 9a5c (strains isolated from orange trees) and Temecula1 (strain isolated from grapevine). We have shown that Fb7 produces up to 57% more OMVs than the 9a5c and Temecula1. Moreover we identified a total of 202 distinct proteins in the OMVs produced by these three strains, increasing considerably the number of OMVs secreted proteins so far described for X. fastidiosa. Among the proteins enriched in OMVs, we point out afimbrial adhesins, porins, lipoproteins, hydrolases (lipases/esterases, proteases and peptidases) and a putative pectin-lyase. We highlight the detection of the enzyme L-ascorbate oxidase in the OMVs and we suggest that this enzyme could act in the depletion of ascorbate produced by the plant host. In addition, we have demonstrated, for the first time, that X. fastidiosa OMVs transport fatty acids from the DSF family, suggesting an additional role for OMVs in this phytopathogen. Finally, in the third study we verified relevant changes in the profile of metabolites secreted by X. fastidiosa in response to the interaction with metabolites secreted by Burkholderia phytofirmans that has been sugested as a biocontrol strain for Pierce's disease in grapevines. We confirm that the B. phytofirmans supernatant has a non-polar compound that induces biofilm formation in X. fastidiosa, but it has not yet been possible to elucidate the chemical nature of this compound

Aplicação exógena do ácido salicílico em maracujazeiro-amarelo para o controle do vírus do endurecimento dos frutos / Exogenous application of salicylic acid in yellow passion fruit for the control of fruit woodiness virus

Foram avaliados o efeito do ácido salicílico (AS) no controle do Cowpea aphid-borne mosaic virus (CABMV), vírus que induz o endurecimento dos frutos do maracujazeiro (EFM), e a sua influência na expressão dos sintomas e na ativação das enzimas peroxidase e polifenoloxidase. O experimento foi delineado e conduzido de forma inteiramente casualizada. Os tratamentos consistiram em AS (2,5 mM) e controle (etanol 10%), aplicados 12 horas antes da inoculação mecânica do CABMV, agente causal do EFM no Brasil. Um experimento similar foi conduzido sob as mesmas condições, porém, quatro aplicações do AS foram realizadas semanalmente após a inoculação mecânica do CABMV. Em ambos os experimentos a avaliação da severidade da doença foi realizada empregando escala de notas que variaram de 0 a 3. Para avaliar a atividade das enzimas peroxidase e polifenoloxidase, as plantas foram tratadas (AS e controle) e, após 12 horas, inoculadas com o isolado de CABMV. Foram realizadas amostragens foliares 0, 12, 24 e 48 horas após os tratamentos (HAT), que foram processadas e analisadas em espectrofotômetro para a constatação da ativação da peroxidase e polifenoloxidase. Aos 30 dias após a inoculação (DAI), o AS aplicado uma única vez promoveu redução da severidade de 57,1%, quando comparado com o controle. Nas plantas submetidas às aplicações semanais de AS foi constatada a redução significativa da expressão dos sintomas aos 45 DAI. Nos ensaios bioquímicos foi observado aumento significativo de peroxidase nos intervalos de 12 horas (DAI)/24 horas (HAT). Para polifenoloxidase foi observado um aumento significativo de sua atividade nos intervalos de 24 horas (DAI)/48 horas (HAT). Sugere-se que o AS pode representar uma ferramenta adicional no manejo do EFM.(AU)

The effect of salicylic acid (SA) was evaluated in control of Cowpea aphid-borne mosaic virus (CABMV), which induces hardening of the fruits of passion fruit (EFM). Also, its influence on the expression of symptoms and the activation of peroxidase and polyphenol oxidases were evaluated. The experiment was designed and conducted in a completely random way. The treatments consisted of SA (2.5 mM) and control (10% ethanol), applied 12 hours before mechanical inoculation of CABMV, the causal agent of EFM in Brazil. A similar experiment was conducted under the same conditions, but four applications were performed weekly after CABMV mechanical inoculation. In both experiments a rating scale ranging from 0 to 3 was used to assess the severity of the disease. To evaluate the activity of peroxidase and polyphenol oxidase, the plants were treated (SA and control) and, after 12 hours, inoculated with isolated CABMV. Leaf samplings were performed at 0, 12, 24 and 48 hours after the treatments (HAT), processed and analyzed in a spectrophotometer to verify the activation of peroxidase and polyphenol oxidase. At 30 days after inoculation (DAI), the SA applied once promoted reduction of 57.1% of the severity, when compared with the control. In plants subjected to SA weekly applications, it was found a significantly reduction in the expression of symptoms at 45 DAI. In the biochemical assays, a significant increase in peroxidase in 12-hour intervals (DAI)/24 hours (HAT) was observed. For polyphenol oxidase, a significant increase of its activity was observed at 24 hour intervals (DAI)/48 hours (HAT). It is suggested that SA may represent an additional tool in the management of EFM.(AU)

Óleo de melaleuca (Melaleuca alternifolia Maiden & Betche, Cheel) no controle de cercosporiose em beterraba / Tea tree oil (Melaleuca alternifolia Maiden& Betche, Cheel) in control of Cercospora beticola Sacc. on Beet

RESUMO O óleo volátil da melaleuca (Melaleuca alternifolia Maiden & Betche, Cheel) possui atividade antimicrobiana podendo causar efeitos sobre as plantas. Avaliou-se a inibição do óleo em Cercospora beticolaSacc., e seu efeito no aumento da produção e qualidade de raízes de beterraba. As doses foram de 0,13; 0,67; 0,80 e 1,00% do óleo, além das testemunhas composta pelo meio de cultura Batata Dextrose Ágar (BDA) no experimento in vitro, e água no experimento in vivo. As plantas foram pulverizadas duas vezes por semana. O delineamento foi inteiramente casualizado, com 4 repetições, e as médias foram comparadas pelo teste Tukey a 5% de probabilidade. O índice de infecção das folhas foi determinado por escala diagramática além do peso e diâmetro das raízes. Os resultados de inibição do crescimento micelial para as doses do óleo foram 0; 56; 87; 83 e 99%, e os índices de infecção: 77,08; 35,62; 21,04; 19,37 e 20,00%, respectivamente, para a testemunha e as doses 0,13; 0,67; 0,80 e 1,00% do óleo. Somente na concentração de 0,80% o óleo proporcionou relação positiva entre o ganho de peso e o diâmetro das raízes. O óleo de Melaleuca foi eficaz no controle de C. beticola e, como consequência, houve produção de raízes de beterraba com melhor desenvolvimento.

ABSTRACT The volatile oil from Melaleuca (Melaleuca alternifolia Maiden & Betche Cheel.) has antimicrobial properties and can promote several effects on plant cultivation. The aim of this study was to evaluate the inhibition of the oil in Cercospora beticola Sacc. and if it favors the growth and development of beet root. The doses were 0.13, 0.67, 0.8 and 1% of oil, besides the control PDA (potato-dextrose-agar) in vitro (laboratory condition) and with water as treatment control in vivo (field conditions). The plants were sprayed twice a week. The treatments were completely randomized and the averages were compared using the Tukey test at 5%. The infection rate of leaves was measured by diagrammatic scale besides the weight and diameter of tubers. The inhibition results of the radial growth by oil treatments were 0; 56, 87, 83 and 99%, while the infection rate showed: 77.08, 35.62, 21.04, 19.37 and 20% respectively to the control and to the oil concentration of 0,13; 0,67; 0,80 e 1,00%. Only at concentration of 0.8% the tea tree oil showed a positive relationship between tuber´s weight and tuber´s diameter gains. It can be concluded that tea tree oil is effective to controlling C. beticola, and also promotes an increase on development in beet tubers.

In Vitro antifungal potency of plant extracts against five phytopathogens

The antifungal activity of aqueous extract of Cannabis sativa, Parthenium hysterophorus, Urtica dioeca, Polystichum squarrosum and Adiantum venustum was investigated against Alternaria solani, Alternaria zinniae, Curvularia lunata, Rhizoctonia solani and Fusarium oxysporum at different concentrations (5, 10, 15 and 20 percent). At 20 percent, maximum antifungal potential was observed with the extracts of C. sativa, which recorded excellent inhibitory activity against C. lunata (100 percent), A. zinniae (59.68 percent), followed by leaf extract of P. hysterophorus (50 percent) against A. solani. The application of botanical extracts for disease management could be less expensive, easily available, non-polluting and eco-friendly.

Efecto del quitosano en el desarrollo in vitro de rhizopus stolonifer ehrenb: fr) vuill en dos medios de cultivo / Effect of chitosan on the in vitro developtment of rhizopus stolonifer (ehrenb: fr) vuill on two culture medium

En este trabajo se evaluó el efecto antifúngico del quitosano (0; 0,5; 1,0; 1,5; 2,0 mg mL-1) en el desarrollo in vitro (crecimiento micelial, formación de cuerpos fructíferos, esporulación, germinación y liberación de proteínas) de Rhizopus stolonifer en dos medios de cultivo (papa dextrosa agar y medio mínimo). Los resultados obtenidos demostraron que el quitosano inhibió el crecimiento micelial de R. stolonifer en ambos medios de cultivo. El mayor índice antifúngico se observó en el medio papa dextrosa agar. El quitosano no afectó la formación de los cuerpos fructíferos de R. stolonifer en los medios estudiados. La esporulación y la germinación de las esporas se afectaron en ambos medios de cultivo por efecto de quitosano, siendo más notable en el medio medio mínimo. Se demostró la liberación de proteínas por efecto del quitosano en medio mínimo y caldo papa dextrosa. En general, en este estudio se evidenció el efecto antifúngico del quitosano en el desarrollo in vitro de Rhizopus stolonifer con independencia del medio de cultivo empleado. Sin embargo, en medio medio mínimo podrían observarse mejor los efectos antifúngicos del quitosano.

In this work the antifungal effect of chitosan (0, 0.5, 1.0, 1.5, 2.0 mg mL-1) on the in vitro development (mycelial growth, fruit bodies formation, sporulation, germination and proteins release) of Rhizopus stolonifer on two culture medium (Potato Dextrose Agar and Minimal medium) was evaluated. The obtained results demonstrated that chitosan inhibited the mycelial growth of R. stolonifer in both culture medium. The highest antifungal effect was observed on potato dextrose agar medium. Chitosan was not affected the fruit bodies formation of R. stolonifer on the studied medium. Sporulation and spore germination were affected in both culture mediun by effect of chitosan, it was more noticeable in minimal medium. It was demonstrated proteins release by effect of chitosan in minimal medium and potato dextrose broth medium. In general, in this study it was showed the antifungal effect of chitosan on in vitro development of Rhizopus stolonifer regardless of the culture medium used. However, in minimal medium could be observed best the antifungal effects of chitosan.

Identificação de genes com expressão modulada por estreptomicina e de genes associados à virulência e patogenicidade em Xylella fastidiosa / Identification of genes modulated by streptomycin and of genes related to virulence and pathogenicity in Xylella fastidiosa

Em concentrações subletais, agentes antimicrobianos modulam a expressão gênica bacteriana, sendo que o conjunto de genes que é modulado depende tanto da cepa bacteriana, como da natureza do agente antimicrobiano. Neste trabalho, avaliamos o perfil de expressão gênica de Xylella fastidiosa cepa 9a5c em resposta ao tratamento por até 60 minutos com dose subletal do antibiótico estreptomicina. Esta é uma cepa virulenta, originalmente isolada de laranjeiras com sintomas de clorose variegada dos citros. A hibridização de microarranjos de DNA representando 2608 das 2848 sequências codificadoras (CDS) previamente anotadas no genoma desta cepa, revelou que 136 CDS apresentaram expressão gênica diferencial em resposta à exposição à estreptomicina, sendo que destas 109 foram negativamente moduladas e 27 positivamente moduladas. Realizamos, também, ensaios de PCR quantitativo precedido de transcrição reversa (RTqPCR) de 21 CDS para confirmar a modulação observada na análise global da expressão gênica. O perfil de expressão gênica de X. fastidiosa em resposta à estreptomicina foi analisado de forma integrada com outros perfis de expressão gênica desta bactéria. Entre as CDS positivamente moduladas, destacamos aquelas codificadoras das chaperoninas GroEL e GroES, que estão associadas a resposta de choque térmico, e CDS associadas à tradução, tais como proteínas ribossomais e fatores de tradução. Interessantemente, a exposição à estreptomicina induz a expressão da CDS que codifica poligalacturonase, que é um fator de virulência em algumas cepas de X. fastidiosa. Por outro lado, o tratamento com estreptomicina promoveu a modulação negativa de CDS relacionadas à formação e manutenção de biofilme ao contrário do observado quando estas bactérias foram submetidas ao tratamento com gomesina, um peptídeo antimicrobiano. O conjunto destas observações sugere que a exposição à dose subletal de estreptomicina possa promover um fenótipo de maior virulência, contrariamente ao efeito...

At sublethal concentrations, antimicrobials compounds modulate bacterial gene expression and the gene set that is modulated depends not only on the bacterial strain but also on the nature of antimicrobial agent. In this study, we evaluated changes in gene expression profile of Xylella fastidiosa strain 9a5c exposed up to 60 min to sublethal concentration of streptomycin. This a virulent strain originally isolated from orange trees with symptoms of citrus variegated chlorosis. Hybridization of DNA microarrays representing 2,608 out of 2848 coding sequences (CDS) previously annotated in strain 9a5c genome revealed 136 CDS differentially expressed upon streptomycin treatment. Of which 109 were down-regulated and 27 up-regulated. Differential expression for a subset of 21 CDS was further evaluated by reverse transcriptionquantitative PCR (RT-qPCR). In addition, we performed an integrated analysis of the gene expression profile of X. fastidiosa in response to streptomycin along with other gene expression profiles available for this bacterium. Among the up-regulated CDS, we highlight those encoding chaperonins GroEL and GroES, which are associated with heat shock response, and those CDS related to translation, such as ribosomal proteins and translation factors. Interestingly, exposure to streptomycin induces the expression of a CDS encoding polygalacturonase, which is a virulence factor for some X. fastidiosa strains. Furthermore, treatment with streptomycin down-regulates some CDS related to biofilm formation oppositely to treatment with gomesin, an antimicrobial peptide. Together, these observations suggest that exposure to sublethal dose of streptomycin might promote a higher virulent phenotype, in contrast to the effect previously observed with gomesin. In the present work, we also describe the pyrosequencing of J1a12 genome, a X. fastidiosa strain that exhibits a less virulent phenotype in citrus and tobacco if compared to strain 9a5c. A comparison of genome...

Comparative analysis of differentially expressed sequence tags of sweet orange and mandarin infected with Xylella fastidiosa

The Citrus ESTs Sequencing Project (CitEST) conducted at Centro APTA Citros Sylvio Moreira/IAC has identified and catalogued ESTs representing a set of citrus genes expressed under relevant stress responses, including diseases such as citrus variegated chlorosis (CVC), caused by Xylella fastidiosa. All sweet orange (Citrus sinensis L. Osb.) varieties are susceptible to X. fastidiosa. On the other hand, mandarins (C. reticulata Blanco) are considered tolerant or resistant to the disease, although the bacterium can be sporadically detected within the trees, but no disease symptoms or economic losses are observed. To study their genetic responses to the presence of X. fastidiosa, we have compared EST libraries of leaf tissue of sweet orange Pêra IAC (highly susceptible cultivar to X. fastidiosa) and mandarin ‘Ponkan’ (tolerant) artificially infected with the bacterium. Using an in silico differential display, 172 genes were found to be significantly differentially expressed in such conditions. Sweet orange presented an increase in expression of photosynthesis related genes that could reveal a strategy to counterbalance a possible lower photosynthetic activity resulting from early effects of the bacterial colonization in affected plants. On the other hand, mandarin showed an active multi-component defense response against the bacterium similar to the non-host resistance pattern.

CitEST libraries

In order to obtain a better understanding of what is citrus, 33 cDNA libraries were constructed from different citrus species and genera. Total RNA was extracted from fruits, leaves, flowers, bark, seeds and roots, and subjected or not to different biotic and abiotic stresses (pathogens and drought) and at several developmental stages. To identify putative promoter sequences, as well as molecular markers that could be useful for breeding programs, one shotgun library was prepared from sweet orange (Citrus sinensis var. Olimpia). In addition, EST libraries were also constructed for a citrus pathogen, the oomycete Phythophthora parasitica in either virulent or avirulent form. A total of 286,559 cDNA clones from citrus were sequenced from their 5’ end, generating 242,790 valid reads of citrus. A total of 9,504 sequences were produced in the shotgun library and the valid reads were assembled using CAP3. In this procedure, we obtained 1,131 contigs and 4,083 singletons. A total of 19,200 cDNA clones from P. parasitica were sequenced, resulting in 16,400 valid reads. The number of ESTs generated in this project is, to our knowledge, the largest citrus sequence database in the world.

The use of RAPD to characterize Bipolaris sorokiniana isolates

Bipolaris sorokiniana is a phytopathogenic fungus causing diseases of cereal crops such as common root rot, the leaf spot disease, seedling blight, and black point of the grain. Random-amplified polymorphic DNA (RAPD) assay was used to investigate the genetic diversity of 20 isolates collected from different cultivars in wheat-producing regions in Brazil. Seventy primers, with random nucleotide sequences, were tested. Reproducibility to amplify the genomic DNA of isolates was found for 30 of the 70 primers tested, generating between 1 and 17 fragments ranging from 0.35 to 2.0 kb (average size). The degree of similarity between samples was calculated through simple association and the dendrogram was assessed using the unweighted pair group method with arithmetical average. After the RAPD analyses 19 isolates were closely grouped, having a similarity coefficient of >or= 78%. Isolate I017 showed very low similarity coefficients, ranging between 38 and 46%. The RAPD analyses provided important information as to the degree of genetic variability and the relationship between the isolates investigated, revealing polymorphism and establishing electrophoretic profiles useful to characterize the phytopathogen.