RESUMEN
The aim of this study was to analyze the effect of essential oils on the control of soft rot of kale. Clove essential oil at 0.25%, lemongrass and palmarosa essential oils at 0.5%, melaleuca and orange essential oils at 0.75%, bergamot, rosemary, sage and ginger essential oils at 1% were evaluated for the in vitro inhibition of Pectobacterium carotovorum subsp. brasiliensis (Pcb) and control of soft rot of kale, sprayed 72 hours before or seven hours after inoculation. Clove, citronella, bergamot, rosemary, palmarosa, sage, melaleuca, and lemongrass oils completely inhibited the growth of Pcb. Lemongrass oil (0.5%) caused 0% of disease incidence (INC), providing 100% of disease control in both periods of inoculation. Clove oil (0.25%) showed a lower INC (25%) when applied after inoculation, providing a control percentage of 71.42%. The lemongrass and clove essential oils were analyzed by GC/FID (Gas Chromatography Flame Ionization Detector) and by GC/MS (Gas Chromatography /Mass Spectrometer). The major components were eugenol (91,9%) for clove oil and citral, isometric mixture of neral (34,1%) and geranial (42,9%) for lemongrass oil. The Minimum inhibitory concentration (MIC) of lemongrass, clove oils and their major components (citral and eugenol, respectively) was determined by using a broth macrodilution technique, as well as they were evaluated at different concentrations on the control of soft rot of kale, sprayed according descriptions above. The MIC was 0.03125% for citral, and 0.0625 and 0.125% for lemongrass and clove oils, respectively. Eugenol didn't show MIC. Lemongrass oil at 0.125% (post-inoculation) and citral at 0.125% (pre and post-inoculation) provided the highest percentages of disease control (33.33, 50, and 100%, respectively). Clove oil at 0.125% (post-inoculation) showed better effectiveness than eugenol (0.25%), providing a percentage of disease control of 16.67%. Lemongrass and clove essential oils were the most effective in control of soft rot of kale, suggesting that these oils have a potential to be used as antibacterial agents.
O objetivo do estudo foi avaliar o efeito de óleos essenciais no controle da podridão mole em couve. Os óleos essenciais de cravo a 0,25%, capim-limão e palmarosa a 0,5%, citronela, melaleuca e laranja a 0,75%, bergamota, alecrim, sálvia e gengibre a 1% foram avaliados na inibição in vitro de Pectobacterium carotovorum subsp. brasiliensis (Pcb) e controle da podridão mole em couve, pulverizados 72 horas antes ou sete horas após a inoculação. Os óleos essenciais de cravo, citronela, bergamota, alecrim, palmarosa, sálvia, melaleuca e capim-limão inibiram completamente o crescimento de Pcb. O óleo de capimlimão (0,5%) promoveu 0% de incidência (INC) da doença (percentual de controle de 100%), em ambos os períodos de inoculação. O óleo de cravo (0,25%) proporcionou menor INC (25%) quando aplicado após inoculação (percentual de controle de 71,42%). Os óleos essenciais de capim-limão e cravo foram analisados por GC/FID (cromatografia gasosa/detector por ionização de chama) e por GC/MS (cromatografia gasosa/ espectometria de massas). Os componentes majoritários foram eugenol (91,9%) no óleo de cravo e citral (neral34,1% e geranial- 42,9%) no óleo de capim-limão. A concentração inibitória mínima (CIM) dos óleos essenciais de capim-limão e cravo e de seus componentes majoritários (citral e eugenol, respectivamente) foi determinada por meio da técnica de macrodiluição em caldo, bem como foram avaliados, em diferentes concentrações, no controle da podridão mole em couve, pulverizados conforme descrito acima. A concentração inibitória mínima (CIM) foi de 0,03125% para o citral, e de 0,0625 e 0,125% para os óleos de capim-limão e cravo, respectivamente. O eugenol não apresentou CIM. O óleo de capim-limão a 0,125% (pós-inoculação) e o citral (0,125%), em ambos os períodos de inoculação, proporcionaram os maiores percentuais de controle (33,33; 50 e 100%, respectivamente). O óleo de cravo a 0,125% (pós-inoculação) mostrou maior eficiência que o eugenol (0,25%), promovendo um percentual de controle de 16,67%. Os óleos essenciais de capim-limão e cravo destacaram-se na eficiência de controle da podridão mole em couve, sugerindo que esses óleos têm potencial para serem utilizados como agentes antibacterianos.
Asunto(s)
Brassica/microbiología , Aceites Volátiles/análisis , Pectobacterium carotovorum/patogenicidad , Plantas/microbiologíaRESUMEN
Iron is an important nutrient for the survival and growth of many organisms. In order to survive, iron uptake from the environment must be strictly regulated and maintained to avoid iron toxicity. The ferric uptake regulator protein (Fur) regulates genes involved in iron homeostasis in many bacteria, including phytopathogens. However, to date, the role played by Fur in the biology of Pectobacterium carotovorum subsp. brasiliense (Pcb1692), an important pathogen of potatoes, has not yet been studied. To this end, we used the lambda recombineering method to generate a fur mutant strain of Pcb1692 and assessed the virulence and fitness of the mutant strain. The results showed that production of siderophores in Pcb1692Δfur increased compared to the Pcb1692 wild-type and the complemented strain Pcb1692Δfur-pfur. However, production of N-acyl homoserine lactone (AHLs), biofilm formation, exopolysaccharide (EPS) production, virulence on potato tubers and swimming motility, were all significantly decreased in Pcb1692Δfur compared to the wild-type and complemented Pcb1692Δfur-pfur strains. The Pcb1692Δfur mutant also demonstrated significant sensitivity to oxidative stress when exposed to H2O2. Consistent with phenotypic results, qRT-PCR results demonstrated that Fur down-regulates genes which encode proteins associated with: iron uptake (HasA-extracellular heme-binding protein and Ferrodoxin-AED-0004132), stress response (SodC-superoxide dismutase), plant cell wall degrading enzymes (PrtA and CelV) and motility (FlhC and MotA). We conclude that the ferric uptake regulator protein (Fur) of Pcb1692 regulates traits that are important to host-pathogens interactions.
Asunto(s)
Proteínas Bacterianas/metabolismo , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidad , Proteínas Represoras/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Regulación hacia Abajo , Interacciones Huésped-Patógeno , Peróxido de Hidrógeno/toxicidad , Hierro/metabolismo , Mutagénesis , Estrés Oxidativo/efectos de los fármacos , Pectobacterium carotovorum/metabolismo , Proteínas Represoras/genética , Sideróforos/metabolismo , Solanum tuberosum/microbiología , Superóxido Dismutasa/metabolismo , Virulencia/genéticaRESUMEN
Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post-harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall-degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild-type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild-type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild-type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI-inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild-type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down-regulated in the mutant compared with the wild-type strain.
Asunto(s)
Mutación/genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/patogenicidad , Enfermedades de las Plantas/microbiología , Tallos de la Planta/microbiología , Percepción de Quorum/genética , Solanum tuberosum/microbiología , Xilema/microbiología , Bioensayo , Susceptibilidad a Enfermedades , Flagelos/metabolismo , Flagelos/ultraestructura , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Pectobacterium carotovorum/ultraestructura , Tubérculos de la Planta/microbiología , Virulencia/genéticaRESUMEN
BACKGROUND: Long noncoding RNAs (lncRNAs) represent a class of RNA molecules that are implicated in regulation of gene expression in both mammals and plants. While much progress has been made in determining the biological functions of lncRNAs in mammals, the functional roles of lncRNAs in plants are still poorly understood. Specifically, the roles of long intergenic nocoding RNAs (lincRNAs) in plant defence responses are yet to be fully explored. RESULTS: In this study, we used strand-specific RNA sequencing to identify 1113 lincRNAs in potato (Solanum tuberosum) from stem tissues. The lincRNAs are expressed from all 12 potato chromosomes and generally smaller in size compared to protein-coding genes. Like in other plants, most potato lincRNAs possess single exons. A time-course RNA-seq analysis between a tolerant and a susceptible potato cultivar showed that 559 lincRNAs are responsive to Pectobacterium carotovorum subsp. brasiliense challenge compared to mock-inoculated controls. Moreover, coexpression analysis revealed that 17 of these lincRNAs are highly associated with 12 potato defence-related genes. CONCLUSIONS: Together, these results suggest that lincRNAs have potential functional roles in potato defence responses. Furthermore, this work provides the first library of potato lincRNAs and a set of novel lincRNAs implicated in potato defences against P. carotovorum subsp. brasiliense, a member of the soft rot Enterobacteriaceae phytopathogens.
Asunto(s)
Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Pectobacterium carotovorum/patogenicidad , ARN Largo no Codificante/genética , ARN de Planta/genética , Solanum tuberosum/genética , Cromosomas de las Plantas/química , Exones , Biblioteca de Genes , Ontología de Genes , Anotación de Secuencia Molecular , Pectobacterium carotovorum/crecimiento & desarrollo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Tallos de la Planta/genética , Tallos de la Planta/inmunología , Tallos de la Planta/microbiología , ARN Largo no Codificante/clasificación , ARN Largo no Codificante/inmunología , ARN de Planta/clasificación , ARN de Planta/inmunología , Solanum tuberosum/inmunología , Solanum tuberosum/microbiologíaRESUMEN
Pectobacterium carotovorum subsp. brasiliense is a newly identified member of the potato soft rot enterobacteriaceae. The pathogenesis of this pathogen is still poorly understood. In this study, an mCherry-P. carotovorum subsp. brasiliense-tagged strain was generated to study P. carotovorum subsp. brasiliense-potato plant interactions. Prior to use, the tagged strain was evaluated for in vitro growth, plasmid stability, and virulence on potato tubers and shown to be similar to the wild type. Four potato cultivars were evaluated for stem-based resistance against P. carotovorum subsp. brasiliense. Confocal laser-scanning microscopy and in vitro viable cell counts showed that P. carotovorum subsp. brasiliense is able to penetrate roots of a susceptible potato cultivar as early as 12 h postinoculation and migrate upward into aerial stem parts. Due to the phenotypic differences observed between tolerant and susceptible cultivars, a comparison of P. carotovorum subsp. brasiliense colonization patterns in these cultivars was undertaken. In the susceptible cultivar, P. carotovorum subsp. brasiliense cells colonized the xylem tissue, forming "biofilm-like" aggregates that led to occlusion of some of the vessels. In contrast, in the tolerant cultivar, P. carotovorum subsp. brasiliense appeared as free-swimming planktonic cells with no specific tissue localization. This suggests that there are resistance mechanisms in the tolerant cultivar that limit aggregation of P. carotovorum subsp. brasiliense in planta and, hence, the lack of symptom development in this cultivar.
Asunto(s)
Resistencia a la Enfermedad , Susceptibilidad a Enfermedades , Pectobacterium carotovorum/patogenicidad , Enfermedades de las Plantas/microbiología , Solanum tuberosum/microbiología , Proteínas Luminiscentes , Microscopía Confocal , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/crecimiento & desarrollo , Fenotipo , Enfermedades de las Plantas/inmunología , Raíces de Plantas/inmunología , Raíces de Plantas/microbiología , Tallos de la Planta/inmunología , Tallos de la Planta/microbiología , Tubérculos de la Planta/inmunología , Tubérculos de la Planta/microbiología , Plásmidos , Proteínas Recombinantes de Fusión , Solanum tuberosum/inmunología , Virulencia , Proteína Fluorescente RojaRESUMEN
Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.
Asunto(s)
Proteínas Anfibias/genética , Péptidos Catiónicos Antimicrobianos/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Solanum tuberosum/genética , Proteínas Anfibias/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacterias/genética , Pollos/genética , Hongos/genética , Regulación de la Expresión Génica de las Plantas , Muramidasa/genética , Muramidasa/metabolismo , Pectobacterium carotovorum/patogenicidad , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Solanum tuberosum/microbiología , Nicotiana/genéticaRESUMEN
The antimicrobial activity of samples of Northern Argentine propolis (Tucumán, Santiago del Estero and Chaco) against phytopathogenic bacteria was assessed and the most active samples were identified. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. Strong antibacterial activity was detected against Erwinia carotovora spp carotovora CECT 225, Pseudomonas syringae pvar tomato CECT 126, Pseudomonas corrugata CECT 124 and Xanthomonas campestris pvar vesicatoria CECT 792. The most active propolis extract (Tucumán, T1) was selected to bioguide isolation and identified for antimicrobial compound (2',4'-dihydroxychalcone). The antibacterial chalcone was more active than the propolis ethanolic extract (MIC values of 0.5-1 µg ml(-1) and 9.5-15 µg ml(-1), respectively). Phytotoxicity assays were realized and the propolis extracts did not retard germination of lettuce seeds or the growth of onion roots. Propolis solutions applied as sprays on tomato fruits infected with P. syringae reduced the severity of disease. Application of the Argentine propolis extracts diluted with water may be promising for the management of post harvest diseases of fruits.
Asunto(s)
Antibacterianos/farmacología , Chalconas/farmacología , Pectobacterium carotovorum/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Própolis/química , Pseudomonas syringae/efectos de los fármacos , Xanthomonas campestris/efectos de los fármacos , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Argentina , Chalconas/química , Chalconas/aislamiento & purificación , Lactuca/efectos de los fármacos , Lactuca/crecimiento & desarrollo , Solanum lycopersicum/microbiología , Pruebas de Sensibilidad Microbiana , Cebollas/efectos de los fármacos , Cebollas/crecimiento & desarrollo , Pectobacterium carotovorum/patogenicidad , Raíces de Plantas/efectos de los fármacos , Pseudomonas syringae/patogenicidad , Semillas/efectos de los fármacos , Xanthomonas campestris/patogenicidadRESUMEN
The natural resistance of plants to disease is based not only on preformed mechanisms, but also on induced mechanisms. The defense mechanisms present in resistant plants may also be found in susceptible ones. This study attempted to analyze the metabolic alterations in plants of the potato Solanum tuberosum L. cv. Agata that were inoculated with the incompatible plant-pathogenic bacteria X. axonopodis and R. solanacearum, and the compatible bacterium E. carotovora. Levels of total phenolic compounds, including the flavonoid group, and the activities of polyphenol oxidase (PPO) and peroxidase (POX) were evaluated. Bacteria compatibility was evaluated by means of infiltration of tubers. The defense response was evaluated in the leaves of the potato plants. Leaves were inoculated depending on their number and location on the stem. Multiple-leaf inoculation was carried out on basal, intermediate, and apical leaves, and single inoculations on intermediate leaves. Leaves inoculated with X. axonopodis and with R. solanacearum showed hypersensitive responses within 24 hours post-inoculation, whereas leaves inoculated with E. carotovora showed disease symptoms. Therefore, the R. solanacearum isolate used in the experiments did not exhibit virulence to this potato cultivar. Regardless of the bacterial treatments, the basal leaves showed higher PPO and POX activities and lower levels of total phenolic compounds and flavonoids, compared to the apical leaves. However, basal and intermediate leaves inoculated with R. solanacearum and X. axonopodis showed increases in total phenolic compounds and flavonoid levels. In general, multiple-leaf inoculation showed the highest levels of total phenolics and flavonoids, whereas the single inoculations resulted in the highest increase in PPO activity. The POX activity showed no significant difference between single- and multiple-leaf inoculations. Plants inoculated with E. carotovora showed no significant increase in defense mechanisms such as enzyme activity and phenolic compounds. Therefore, resistance or susceptibility in S. tuberosum cv. Agata might depend on leaf age, type of inoculation performed (single or multiple), and the interaction between plant and pathogen.
Asunto(s)
Pectobacterium carotovorum/fisiología , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/fisiología , Solanum tuberosum/microbiología , Xanthomonas axonopodis/fisiología , Interacciones Huésped-Parásitos , Inmunidad Innata/fisiología , Pectobacterium carotovorum/patogenicidad , Enfermedades de las Plantas/inmunología , Ralstonia solanacearum/patogenicidad , Solanum tuberosum/enzimología , Solanum tuberosum/inmunología , Xanthomonas axonopodis/patogenicidadRESUMEN
The natural resistance of plants to disease is based not only on preformed mechanisms, but also on induced mechanisms. The defense mechanisms present in resistant plants may also be found in susceptible ones. This study attempted to analyze the metabolic alterations in plants of the potato Solanum tuberosum L. cv. Agata that were inoculated with the incompatible plant-pathogenic bacteria X. axonopodis and R. solanacearum, and the compatible bacterium E. carotovora. Levels of total phenolic compounds, including the flavonoid group, and the activities of polyphenol oxidase (PPO) and peroxidase (POX) were evaluated. Bacteria compatibility was evaluated by means of infiltration of tubers. The defense response was evaluated in the leaves of the potato plants. Leaves were inoculated depending on their number and location on the stem. Multiple-leaf inoculation was carried out on basal, intermediate, and apical leaves, and single inoculations on intermediate leaves. Leaves inoculated with X. axonopodis and with R. solanacearum showed hypersensitive responses within 24 hours post-inoculation, whereas leaves inoculated with E. carotovora showed disease symptoms. Therefore, the R. solanacearum isolate used in the experiments did not exhibit virulence to this potato cultivar. Regardless of the bacterial treatments, the basal leaves showed higher PPO and POX activities and lower levels of total phenolic compounds and flavonoids, compared to the apical leaves. However, basal and intermediate leaves inoculated with R. solanacearum and X. axonopodis showed increases in total phenolic compounds and flavonoid levels. In general, multiple-leaf inoculation showed the highest levels of total phenolics and flavonoids, whereas the single inoculations resulted in the highest increase in PPO activity. The POX activity showed no significant difference between single- and multiple-leaf inoculations. Plants inoculated with E. carotovora showed no significant increase ...