Ricardo Magela de Souza

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.

Rhizobacterium and acibenzolar-S-methyl (ASM) in resistance induction against bacterial blight and expression of defense responses in cotton

The effect of acibenzolar-S-methyl (ASM) and the rhizobacterium isolate L2-1 (Bacillus cereus), applied either alone or in combination, was evaluated in cotton plants for resistance induction against Xanthomonas axonopodis pv. malvacearum. Bacterial blight severity, lignin content and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX) and b-1.3 glucanase (GLU) were assessed at different time courses. ASM and L2-1, used alone or in combination, significantly reduced disease severity, while ASM alone was the best treatment for bacterial blight control. No synergistic effect was observed when ASM was associated to isolate L2-1. ASM induced the highest increases in the activities of PAL, POX and GLU as well as lignin deposition, mainly 14 days after treatment application. When ASM and the rhizobacterium were simultaneously applied, enzyme activities were always lower, compared to ASM alone.

Transcriptional profiling in cotton associated with Bacillus subtilis (UFLA285) induced biotic-stress tolerance

Plant growth promoting rhizobacteria (PGPR) confer disease resistance in many agricultural crops. In the case of Bacillus subtilis (UFLA285) isolated from the cotton producing state of Mato Grosso (Brazil), in addition to inducing foliar and root growth, disease resistance against damping-off caused by Rhizoctonia solani was observed. The aim of this cotton study was to identify gene transcriptional events altered with exposure to the PGPR strain UFLA285 in infected plants. Global gene transcription was profiled using a commercially-available cotton gene chip; cotton plants with and without UFLA285-seed treatment were infected with R. solani 9-days after planting and harvested on day14. Microarray data of stem tissue revealed 247 genes differentially regulated in infected plants, seed treated versus untreated with UFLA285. Transcripts encoding disease resistance proteins via jasmonate/ethylene signaling as well as osmotic regulation via proline synthesis genes were differentially expressed with UFLA285 induction. Consistent with transcriptional regulation, UFLA285 increased plant-proline accumulation and dry weight. This study has identified transcriptional changes in cotton, induced by the beneficial soil bacterium UFLA285 and associated with disease control.

Antibacterial activity of essential oils on Xanthomonas vesicatoria and control of bacterial spot in tomato

The objective of this work was to evaluate the effects of plant essential oils (EOs) on the growth of Xanthomonas vesicatoria, on bacterial morphology and ultrastructure, and on the severity of tomato bacterial spot. EOs from citronella, clove, cinnamon, lemongrass, eucalyptus, thyme, and tea tree were evaluated in vitro at concentrations of 0.1, 1.0, 10, and 100% in 1.0% powdered milk. The effect of EOs, at 0.1%, on the severity of tomato bacterial spot was evaluated in tomato seedlings under greenhouse conditions. The effects of citronella, lemongrass, clove, and tea tree EOs, at 0.1%, on X. vesicatoria cells were evaluated by transmission electron microscopy. All EOs showed direct toxic effect on the bacteria at a 10%-concentration in vitro. Under greenhouse conditions, the EOs of clove, citronella, tea tree, and lemongrass reduced disease severity. EOs of clove and tea tree, and streptomycin sulfate promoted loss of electron-dense material and alterations in the cytoplasm, whereas EO of tea tree promoted cytoplasm vacuolation, and those of citronella, lemongrass, clove, and tea tree caused damage to the bacterial cell wall. The EOs at a concentration of 0.1% reduce the severity of the disease. Resumo - O objetivo deste trabalho foi avaliar o efeito de oleos essenciais (OEs) no crescimento de Xanthomonas vesicatoria, na morfologia e na ultraestrutura bacteriana, e na severidade da mancha-bacteriana do tomateiro. OEs de citronela, cravo-da-india, canela, capim-limao, eucalipto, tomilho e arvore-de-cha foram avaliados in vitro nas concentracoes de 0,1, 1,0, 10 e 100% em leite em po a 1,0%. O efeito dos OEs, a 0,1%, na severidade da mancha-bacteriana do tomateiro foram avaliados em plantas de tomateiro em casa de vegetacao. Os efeitos dos oleos de citronela, capim-limao, cravo-da-india e arvore-de-cha, a 0,1%, nas celulas de X. vesicatoria foram avaliados por meio de microscopia eletronica de transmissao. Todos os OEs apresentaram efeito toxico direto sobre as bacterias na concentracao de 10% in vitro. Em casa de vegetacao, os OEs de cravo-da-india, citronela, arvore-de-cha e capim-limao reduziram a severidade da doenca. Os OEs de cravo-da-india e arvore-de-cha, e o sulfato de estreptomicina promoveram perda de material eletro-denso e alteracoes no citoplasma, enquanto o OE de arvore-de-cha promoveu vacuolizacao do citoplasma, e os de citronela, capim-limao, cravo-da-india e arvore-de-cha causaram danos a parede celular bacteriana. Os OEs, na concentracao de 0,1%, reduzem a severidade da doenca. Termos para indexacao: controle alternativo de doencas de plantas, ultraestrutura celular, bacteria fitopatogenica.

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 103-107 CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 103-106 CFU/mL and immuno-FCM from 104-106 CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds.

Bactérias endofíticas como agentes promotores do crescimento de plantas de tomateiro e de inibição in vitro de Ralstonia solanacearum

A partir de 150 isolados de bacterias endofiticas obtidos de folhas, caules e raizes de tomateiros sadios, 53 destacaram-se quanto a habilidade em promover o crescimento de plantas de tomateiro (Solanum lycopersicum L.). Submetidos a uma nova selecao, os isolados UFV-E17, UFV-E22, UFV-E25, UFV-E26, UFV-E27, Bacillus cereus (UFV-E29), UFV-E49, UFLA 06-LS, UFLA 08-LS e UFLA 11-LS apresentaram maior promocao do crescimento. Avaliacoes semanais de altura e numero de folhas e foliolos das plantas aconteceram durante 45 dias. Apos a sexta avaliacao, mensurou-se a area foliar e o peso da materia fresca e seca da parte aerea e da raiz das plantas. O isolado UFV-E49 apresentou melhor resultado para altura, area foliar, numero de folhas e peso da materia fresca e seca, tanto da parte aerea quanto da raiz. Dos isolados selecionados, somente dois apresentaram efeito inibitorio direto in vitro a Ralstonia solanacearum.

Aumento da eficiência nutricional de tomateiros inoculados com bactérias endofíticas promotoras de crescimento

Plant growth-promoting endophytic bacteria can increase plant nutritional efficiency thus favouring its yield. With the purpose of evaluating the influence of 10 previously selected isolates of growth-promoting endophytic bacteria on the uptake, utilization and transport of nutrients by tomato plants, greenhouse experiments were installed. The hypocotyl was cut in order to apply the endophytic bacteria to tomato seedlings cultivar Santa Clara. Fifty five days after transplanting the upper portion of the cut seedlings, the plants were collected to determine the dry matter of the aerial parts and concentration of macro and micro nutrients. The concentration of N, P, K, Ca, Mg, Cu and Zn in the shoot and N, P, Mg and Mn in roots of inoculated plants differed from non-inoculated controls. Endophytic bacteria Micrococcus sp. (UFLA 11-LS) and Brevundimonas sp. (UFV-E49) were identified by sequencing of the 16S ribosomal DNA. The P uptake in plants inoculated with these isolates was higher than in the non-inoculated controls. Plants treated with the first isolate were more efficient in the use of N, P, K, Ca, Mg, S, Cu, Fe, and Zn. The highest concentration of N, P, K, Mg, and Zn were found in the shoot of plants inoculated with Brevundimonas sp. The results of this study indicate that these endophytic bacteria isolates may be employed to increase the nutritional efficiency of tomato plants.

Filtrados de culturas bacterianas endofíticas na motilidade, mortalidade e eclosão de juvenis de segundo estádio de Meloidogyne javanica

To obtaining the filtrates, bacteria were cultivated in liquid trypic soy broth medium for seven days at 28 oC under constant stirring, centrifugated at 10,000 g for 15 min and the supernatant was filtrated by 0,22 µm Millipore. Nonmotile Meloidogyne javanica and dead ones were counted 24 and 48 h later and hatched J2 were counted 15 days after setting up the experiment. Seven of 40 bacterial isolates tested immobilized the juveniles within 24 h, and they could not recover even in water. This effect was similar to the aldicarb treatment used as control. The filtrates of isolates also inhibited egg hatching. Two isolates killed more than 90% of J2 after 48 h of exposure. Filtrate dilutions of the most efficient isolate in 1:1 and 1:2 (bacterial filtrate: water) also showed to have effective action on nematode behaviors.

Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão em meio de cultura semi seletivo

To detect Xanthomonas axonopodis pv. phaseoli (Xap) in whole and crushed bean seed extracts, naturally contaminated and healthy bean seeds were used in the semi-selective media XCP1 and MT and in the non-selective medium 523. Seed extracts were also inoculated in the first leaves of bean plants. The semi-selective medium XCP1 was more efficient in the quantification and detection of Xap in whole bean seed extracts. Naturally contaminated seed extracts, inoculated in the first leaves of bean plants, showed the symptoms of bacterial blight.

Induced defense responses in tomato against bacterial spot by proteins synthesized by endophytic bacteria

Some endophytes can synthesize molecules that elicit the induction of plant resistance to infection by pathogens. The objective of this study was to demonstrate that protein fractions 42 and 75 from Bacillus amyloliquefaciens and Bacillus pumilus were capable of acting as elicitors of induced resistance in tomato plants against Xanthomonas vesicatoria, following partial resolution by gel-filtration chromatography. Tomato plants sprayed with protein fractions 42 and 75 reduced, respectively, 63.5 and 56.6% of bacterial spot, compared with control plants. Additionally, these fractions promoted the increase of peroxidase (POX) and polyphenol oxidase (PPO) enzyme activities in treated plants. In SDS-PAGE stained with silver nitrate, protein fractions 42 and 75 appeared as simple bands with estimated molecular mass of 28 and 43 kDa, respectively. We report the partial characterization of two macromolecules synthesized by endophytic bacteria that act as elicitors of systemic resistance in tomato against X. vesicatoria.