RESUMO
Ralstonia solanacearum species complex (RSSC) is a phytopathogenic bacterial group that causes bacterial wilt in several crops, being potato (Solanum tuberosum) one of the most important hosts. The relationship between the potato plant ionome (mineral and trace elements composition) and the resistance levels to this pathogen has not been addressed until now. Mineral content of xylem sap, roots, stems and leaves of potato genotypes with different levels of resistance to bacterial wilt was assessed in this work, revealing a positive correlation between divalent calcium (Ca) cation concentrations and genotype resistance. The aim of this study was to investigate the effect of Ca on bacterial wilt resistance, and on the growth and virulence of RSSC. Ca supplementation significantly decreased the growth rate of Ralstonia pseudosolanacearum GMI1000 in minimal medium and affected several virulence traits such as biofilm formation and twitching motility. We also incorporate for the first time the use of microfluidic chambers to follow the pathogen growth and biofilm formation in conditions mimicking the plant vascular system. By using this approach, a reduction in biofilm formation was observed when both, rich and minimal media, were supplemented with Ca. Assessment of the effect of Ca amendments on bacterial wilt progress in potato genotypes revealed a significant delay in disease progress, or a complete absence of wilting symptoms in the case of partially resistant genotypes. This work contributes to the understanding of Ca effect on virulence of this important pathogen and provides new strategies for an integrated control of bacterial wilt on potato. IMPORTANCE: Ralstonia solanacearum species complex (RSSC) includes a diverse group of bacterial strains that cause bacterial wilt. This disease is difficult to control due to pathogen aggressiveness, persistence, wide range of hosts, and wide geographic distribution in tropical, subtropical, and temperate regions. RSSC causes considerable losses depending on the pathogen strain, host, soil type, environmental conditions, and cultural practices. In potato, losses of $19 billion per year have been estimated for this pathogen worldwide. In this study, we report for the first time the mineral composition found in xylem sap and plant tissues of potato germplasm with different levels of resistance to bacterial wilt. This study underscores the crucial role of calcium (Ca) concentration in the xylem sap and stem in relation to the resistance of different genotypes. Our in vitro experiments provide evidence of Ca's inhibitory effect on the growth, biofilm formation, and twitching movement of the model RSSC strain R. pseudosolanacearum GMI1000. This study introduces a novel element, the Ca concentration, which should be included into the integrated disease control management strategies for bacterial wilt in potatoes.
Assuntos
Cálcio , Doenças das Plantas , Ralstonia solanacearum , Solanum tuberosum , Solanum tuberosum/microbiologia , Doenças das Plantas/microbiologia , Cálcio/metabolismo , Ralstonia solanacearum/fisiologia , Ralstonia solanacearum/genética , Ralstonia solanacearum/patogenicidade , Ralstonia solanacearum/crescimento & desenvolvimento , Virulência , Biofilmes/crescimento & desenvolvimento , Ralstonia/genética , Ralstonia/fisiologia , Raízes de Plantas/microbiologia , Xilema/microbiologiaRESUMO
The intracellular accumulation of polyhydroxyalkanoates (PHAs) normally occurs after cell growth, during the second fermentation stage and under nutrient-limited conditions in the presence of a carbon excess. However, some microorganisms are able to accumulate PHAs as poly(3-hydroxybutyrate) [P(3HB)] during the first fermentation stage, the cell growth phase, without nutrient limitation, once they have been reported to utilize type II metabolism during the polymer accumulation phase. This study evaluated the effect of aeration and agitation on cell growth and P(3HB) accumulation in Ralstonia solanacearum RS, performed in a bioreactor for 24h at 32°C. A 22 central composite rotational design (CCRD) was used, with agitation (150 to 250 rpm) and aeration (0.3 to 1 vvm) as independent variables and optical density (OD600nm), dry cell weight (DCW), and P(3HB) yield as dependent variables. A significant polymer accumulation, until 70% of P(3HB), was observed, proving that R. solanacearum RS exhibited metabolism type II, regardless of the aeration process. The best results were obtained for 1 vvm and 250 rpm (+1, +1), with values of OD600nm (18.04) and DCW (4.82 g.L-1).
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Ralstonia solanacearum/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Meios de Cultura/metabolismo , Fermentação , Microbiologia Industrial , Ralstonia solanacearum/metabolismo , RotaçãoRESUMO
Poly(3-hydroxybutyrate) (P(3HB)) is a biodegradable plastic biopolymer that accumulates as lipophilic inclusions in the cytoplasm of some microorganisms. The biotechnological process by which P(3HB) is synthesized occurs in two phases. The first phase involves cell growth in a complex culture medium, while the second phase involves polymer accumulation in the presence of excess carbon sources. As such, the efficiency of the second phase depends on the first phase. The aim of this study was to evaluate culture media with different concentrations of sucrose and glucose and different pH values in the inoculum phase of Ralstonia solanacearum RS with the intention of identifying methods by which the biomass yield could be increased, subsequently enhancing the yield of P(3HB). The culture medium was formulated according to the experimental planning type of central composite rotational design 22. The independent variables were pH and sugar concentration (sucrose and glucose), and the dependent variables were OD600nm, dry cell weight (DCW), and P(3HB) yield. The highest cell growth, estimated by the OD600nm (20.6) and DCW (5.35) values, was obtained when sucrose was used in the culture medium at a concentration above 35 g.L-1 in combination with an acidic pH. High polymer (45%) accumulation was also achieved under these conditions. Using glucose, the best results for OD600nm (12.5) and DCW (2.74) were also obtained at acidic pH but with a sugar concentration at the minimum values evaluated. Due to the significant accumulation of polymer in the cells that were still in the growth phase, the accumulating microorganism P(3HB) Ralstonia solanacearum RS can be classified as having type II metabolism in relation to the polymer accumulation phase, which is different from other Ralstonia spp. studied until this time.
Assuntos
Glucose/metabolismo , Microbiologia Industrial/métodos , Modelos Teóricos , Poliésteres/metabolismo , Ralstonia solanacearum/metabolismo , Biomassa , Biotransformação , Meios de Cultura/química , Fermentação , Glucose/análise , Concentração de Íons de Hidrogênio , Ralstonia solanacearum/crescimento & desenvolvimentoRESUMO
Tobacco germplasm samples with various levels of resistance to bacterial wilt were selected to construct F1 combinations of parental inbred lines and orthogonal diallel crosses using samples collected in 2009 (15 germplasms), 2010 (15 germplasms), and 2011 (16 germplasms). A total of 1/2P (P + 1) experimental materials were used for analysis. Based on the analyses of major and minor locus groups, genetic effects on the incidence rate and index of bacterial wilt in tobacco were investigated on the 15th and 25th day during the early stage. Significant effects were observed in major locus groups, but not in minor locus groups. Specifically, adjacent major locus groups (J1 = 13,056 and J1 = 13,055; J1 = 14,080 and J1 = 14,079) were detected in both the first and second analyses with considerable effects. Based on the additive effects of minor locus groups on the rate and index of bacterial wilt, the effects on the incidence rates of Yunyan 85, DB101, and RG11 as well as the effects on the disease index of the latter two germplasms reached the maximum. This was consistent with the disease resistance indicators of these tobacco varieties in the field (corresponding broad heritability >20%). Genetic homozygous dominant loci (+ +) increased the rate of bacterial wilt (susceptible), whereas homozygous recessive loci (- -) reduced the index of bacterial wilt (resistant) with considerable additive effects and low dominant effects, suggesting that the inheritance of the bacterial wilt rate and index in tobacco mainly relies on additive inheritance.
Assuntos
Resistência à Doença/genética , Nicotiana/genética , Doenças das Plantas/genética , Locos de Características Quantitativas , Alelos , Resistência à Doença/imunologia , Ligação Genética , Homozigoto , Padrões de Herança , Modelos Genéticos , Doenças das Plantas/imunologia , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/patogenicidade , Banco de Sementes , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
Screening is a critical step in the discovery of microbial agents that can exert biological control of Fusarium verticillioides at the root level. The objectives of this research were to determine the utility of a niche overlap index to realise the first screening of maize rhizobacterial isolates during different water activities. Studies were conducted to evaluate various methods for second screening with different modes of action. The antifungal activity of bacterial isolates through antibiosis assay was checked and the influence of different isolates on Fusarium verticilliodes growth and fumonisin B(1) was studied. Eleven competitive rhizobacterial isolates (Arthrobacter globiformis RC1, Azotobacter armeniacus RC2, A. armeniacus RC3, A. globiformis RC4, A. globiformis RC5, A. armeniacus RC6, Pseudomonas solanacearum RC7, Bacillus subtilis RC8, B. subtilis RC9, P. solanacearum RC10, B. subtilis RC11) were selected for the studies which followed. All bacteria were able to utilise the widest range of carbon sources and showed the highest niche overlap indices at the water activities tested. All bacterial antagonists reduced fumonisin B(1) production at all levels tested. Isolates belonging to Pseudomonas and Bacillus genera significantly inhibited fumonisin B(1) production, which ranged between 70 and 100%. Also, A. armeniacus RC2 caused important fumonisin B(1) reduction. The results of the present work suggest that A. armeniacus RC2, A. armeniacus RC3, B. subtilis RC8, B. subtilis RC9, B. subtilis RC11, P. solanacearum RC7, and P. solanacearum RC10 could have practical value in the control of F. verticillioides root colonisation. This paper is part of an on-going study to determine their application at the field level.