Ana Beatriz Zacaroni

Acibenzolar-S-Metil e Ecolife® na indução de respostas de defesa do tomateiro contra a mancha bacteriana (Xanthomonas vesicatoria)

Foi investigada a eficacia comparativa da pulverizacao foliar em tomateiro de acibenzolar-S-metil (ASM) e Ecolife® na protecao contra Xanthomonas vesicatoria, bem como avaliada a ativacao de algumas respostas bioquimicas de defesa de planta. Plantas de tomateiro cv. Santa Cruz Kada foram pulverizadas com acibenzolar S-metil (0,2 g l-1 ASM) e uma formulacao natural proveniente de biomassa citrica denominada Ecolife® (5 ml l-1). Quatro dias apos as pulverizacoes, as plantas foram inoculadas com um isolado patogenico de Xanthomonas vesicatoria. Em experimentos de quantificacao de doenca, a pulverizacao foliar de Ecolife® e ASM conferiu 39,2% e 47,7% de protecao, respectivamente. A resistencia induzida em plantas pulverizadas com ASM e Ecolife® foi evidenciada pelo aumento da atividade de peroxidases (POX) e oxidases de polifenois (PPO), iniciado logo as primeiras horas apos as pulverizacoes, continuando ate 12 dias de avaliacao. A despeito da tendencia de queda nas atividades de amonia-liases de fenilalanina (PAL) a partir de 3 dias apos as pulverizacoes, plantas tratadas com ASM e Ecolife® tiveram discreto aumento no acumulo de lignina, principalmente aquelas pulverizadas com Ecolife® e inoculadas com X. vesicatoria. Teores de fenois soluveis totais decresceram significativamente, 9 e 12 dias apos pulverizacoes. O aumento nas atividades de POX e PPO poderia resultar em lignificacao, a qual estaria associada a uma estrategia de defesa do tomateiro contra a mancha bacteriana.

Óleo essencial de Lippia sidoides nativas de Minas Gerais: composição, estruturas secretoras e atividade antibacteriana

Given the diverse biological activities presented by essential oils, and the antibacterial potential of the essential oil of Lippia sidoides Cham, the objective of this work was to characterise the essential oil of this species, collected in the southern part of the state of Minas Gerais, Brazil, as well as to identify its secretory structures and evaluate its antimicrobial activity on the phytobacteria, Clavibacter michiganensis subsp. Michiganensis, Xanthomonas vesicatoria and Pseudomonas syringae pv. tomato. Plants were collected in the town of Itumirim, and the essential oils of the leaves extracted by hydrodistillation using a modified Clevenger apparatus. The essential oil presented carvacrol (26.44%) and 1.8-cineole (22.63%) as major constituents. On the leaf surface of L. sidoides both glandular and non-glandular trichomes were found. Among the glandular trichomes are peltate trichomes and type I capitate trichomes. The presence of lipidic and terpenic substances within the head cells of both the peltate and capitate trichomes was found. The essential oil of L. sidoides was able to inhibit the growth of all the microorganisms studied, showing the following MIC: 7.81 µg mL-1 for C. michiganensis, 3.9 µg mL-1 for P. syringae and 15.62 µg mL-1 for X. vesicatoria. Carvacrol, the major constituent, showed MIC values equal to 3.9 µg mL-1 for all three bacteria studied. Both exhibited the ability to inhibit the growth of gram-positive and gram-negative bacteria.

Otimização da técnica de PCR para a detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão

O crestamento bacteriano comum, causado por Xanthomonas axonopodis pv. phaseoli (Xap), e a principal bacteriose do feijoeiro no Brasil, sendo transmitida principalmente por sementes. O presente trabalho teve como objetivo aperfeicoar uma tecnica, por meio de diferentes metodos de preparacao do extrato, para a deteccao de Xap, bem como sua deteccao simultânea com Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) nos extratos de sementes de feijao,via PCR. A partir de amostras de sementes de feijao inoculadas artificialmente com Xap e lotes comerciais, foram avaliados: extrato bruto obtido diretamente das sementes, extrato concentrado por filtracao em membrana de 0,22µM de diâmetro, extrato concentrado por centrifugacao e extrato plaqueado em meio semi-seletivo XCP1 com e sem antibioticos (BIO-PCR). Avaliou-se a presenca simultânea de Xap e Cff em 10 lotes comerciais de sementes de feijao atraves da reacao multiplex, utilizandos-se os primers X4c, X4e, CffFOR2 e CffREV4. A partir do extrato bruto, do extrato concentrado por centrifugacao e por filtragem em membrana Millipore® nao foi possivel a deteccao de Xap nas sementes de feijao artificialmente contaminadas nem nos 47 lotes comerciais de sementes/ graos de feijao. A tecnica de BIO-PCR permitiu a deteccao de Xap a partir de extratos de sementes de feijao artificialmente contaminadas e em 18 dos 47 lotes comerciais. A tecnica de deteccao simultânea de Xap e Cff no mesmo gel e viavel, por amplificar fragmentos de DNA tipicos de cada fitobacteria. O uso do meio de cultura XCP1 sem adicao de antibioticos permitiu detectar Xap com periodo de incubacao menor em um dia em comparacao a deteccao utilizando-se o meio de cultura com antibioticos

Sensibilidade do método de obtenção das células bacterianas e da técnica de PCR para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em sementes de feijão

Currently, there is a need to develop sensitive methods, inexpensive, reproducible and rapid detection of phytobacteria seeds. The objective of this study was to optimize a method for obtaining bacterial cells and the PCR technique to detect Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), in bean seeds using PCR. It was used one pairs of primers CffFOR2-REV4, designed from an PCR amplified fragment of the conserved repetitive sequence (Rep-PCR). The primer pair CffFOR2-REV4 was selected in this study, because demonstrated higher reproducibility and efficacy in the detection of Cff strains in bean seeds. Four methods were also evaluated in the seed extract preparation for PCR: 1) rough seed extract; 2) millipore membrane filter (0.22Mu diameter) concentrated seed extract and subsequently ressuspended in water; 3) centrifuge concentrated seed extract 10.000 xg for 15 minutes for 20 and 80 mL; 4) Bio-PCR. Among those methods, either Bio-PCR or centrifuge concentrated extract, in a total 20 or 80 mL suspension volume, produced a 306bp DNA fragment, diagnostic for Cff. Those two techniques detected the bacterium and presented high sensitivity, detecting up to 1 Cff physiological conditioning artificially contaminated seed in a total 999 healthy ones. A total of seventeen commercial bean seed lots were analyzed by the method of concentration of the extract by centrifugation, and twelve detected in the bacterium Cff, observed by the presence of bands with 306 bp. Therefore, it was possible to optimize a method of obtaining the bacterial cells and PCR for detection of Cff in bean seeds, that is sensitive, reproducible and easy to perform, which can be used routinely in laboratories for seed.

Inoculação de Curtobacterium flaccumfaciens pv. flaccumfaciens em sementes de feijão por meio da técnica de condicionamento fisiológico

This study used the physiological conditioning technique in an agar-based medium for Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) to inoculate dry beans. The seed performance of the Perola dry bean variety was evaluated after submitting it to physiological conditioning in a water restricted agar-based medium. The treatments consisted of exposing dry bean seeds to the bacteria for different time periods at four levels of water restriction in 523 medium (-0.55, -0.75, -0.95 and -1.15 MPa), and four substrates (523 medium, 523 medium+KCl, 523 medium+mannitol and 523 medium+sucrose) with an additional control treatment of 523 medium without any osmotic restriction (-0,55 MPa). After each time period, the number of germinated seeds in the medium was evaluated and the water content, germination and seed enzymatic patterns were assessed. In a second study, growth was assessed for four Cff strains (Cff DF - Feij-2936, Cff PR - 12768, Cff SC - Feij-2928 e Cff SP - Feij-2634) in different water restriction 523 media. The treatments in the third phase were defined on the basis of the results of the first stage, where the best combination was mannitol-amended 523 medium set for -0,95 MPa water potential and 48 hours exposure to the pathogen. In the second phase, the Cff SC (Feij-2928) strain from Santa Catarina state showed the best growth in an agar-based medium and water potential as defined in the first phase. Thus, Cff artificial inoculation in bean seeds was done by using the physiological conditioning technique in an agar-based medium without the occurrence of any detectable changes in the seed physiological quality patterns.

Bacterial Leaf Spot of Radicchio (Cichorium intybus) is caused by Xanthomonas hortorum

Radicchio (Cichorium intybus) is ranked 22 among crops in Monterey County, California, with a farm gate value of

Rizobactérias no controle da mancha angular do algodoeiro

19,531,000 (3). Beginning in 2002, a leaf spot disease of radicchio was observed in Monterey County. The disease began as small lesions and in some cases coalesced into larger, irregular spots. Lesions were maroon to dark brown; in some cases, the margins of brown lesions became dark maroon with aging. Each leaf spot was observable from both adaxial and abaxial leaf surfaces. Symptoms primarily occurred on the outer foliage of the heads, though on occasion the head cap leaf could develop lesions. Disease incidence in the first year resulted in up to 10% unharvested radicchio because of cap leaf infections or reduced head size if outer wrapper leaves were all removed; outbreaks in subsequent seasons were more limited. Bacteria forming yellow mucoid colonies were isolated from surface disinfested symptomatic tissue that was macerated and streaked onto sucrose peptone agar medium. Bacteria were gram negative, did not fluoresce on Kings Medium B, and used esculin as a carbon source but used none of the other 48 carbon sources tested using the API 50 CH test strip. Nine isolates from symptomatic radicchio had the same DNA fragment banding pattern generated by repetitive extragenic palindromic sequence polymerase chain reactions (rep-PCR) using the BOXA1R primer. Amplicons of rpoD, dnaK, fyuA, and gyrB for multilocus sequence typing (MLST) were generated using a modification of the scheme developed by Young et al. (4) and sequenced by a commercial laboratory. Concatenated sequences of the four genes from the radicchio isolates were compared to the sequences available in the Plant Associated and Environmental Microbes Database (1). The genetic distance between the nine isolates from radicchio and pathotypes of Xanthomonas hortorum were 0.03 or less and MLST analysis indicated that radicchio isolates were members of the species X. hortorum (2). To complete Kochs postulates, freshly grown cultures were suspended in phosphate buffer and adjusted to approximately 5 × 108 CFU/ml. The inoculum was sprayed onto the undersides of leaves of 40-day-old radicchio plants (C. intybus cv. Leonardo). Plants were incubated at 100% humidity for 48 h and then moved to a greenhouse. Plants sprayed with buffer served as negative controls. For each of the two experiments conducted, there were three and six single-plant replicates per treatment. The buffer treated plants did not develop symptoms but the plants treated with isolates from radicchio developed leaf spots similar to those observed in the field with symptoms beginning to be visible after 5 days. The bacteria isolated from symptomatic tissue on inoculated plants were identical to the original strains when compared with rep-PCR, thus completing Kochs postulates. Results from the two experiments were similar. To our knowledge, this is the first report of X. hortorum causing a leaf spot disease on radicchio. The disease continues to occur sporadically on radicchio grown in coastal California. References: (1) Almeida et al. Phytopathology 100:208, 2010. (2) Bull et al. Phytopathology 101:847, 2011. (3) Lauritzen, Monterey County Crop Report, 2010; (4) Young et al. Syst. Appl. Microbiol. 31:366, 2008.

INDIAN CLOVE ESSENTIAL OIL IN THE CONTROL OF TOMATO BACTERIAL SPOT

Avaliou-se o potencial de rizobacterias na inducao de resistencia do algodoeiro a Xanthomonas axonopodis pv. malvacearum. Apos o isolamento das rizobacterias, foram selecionados os isolados capazes de reduzir os sintomas da mancha angular bacteriana em casa de vegetacao, os quais foram aplicados espacialmente separados do patogeno desafiador. Os melhores isolados foram testados quanto a capacidade de reduzir os sintomas da ramulose e da murcha de Verticillium e de inibir diretamente os patogenos in vitro. Do total de 123 isolados de rizobacterias 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%, em relacao a testemunha. Nenhum isolado reduziu a severidade da ramulose e da murcha de Verticillium em relacao a testemunha, nem apresentou efeito inibitorio direto in vitro a X. axonopodis pv. malvacearum e Colletotrichum gossypii var. cephalosporioides. Para V. dahliae, apenas o isolado L2-1 apresentou efeito inibitorio.