Abstract: Silicon amendment significantly reduced bacterial wilt incidence expressed as area under disease progress curve for tomato genotypes L390 (susceptible) by 26.8% and King Kong2 (moderately resistant) by 56.1% compared to non-treated plants grown in hydroponic culture. However, wilt incidence in silicon-treated plants of genotype L390 reached 100% at 13 days post-inoculation (dpi), while in genotype King Kong2, plant death was retarded by 6 days, with 20% reduction of final wilt incidence. Bacterial numbers were significantly lower in silicon-treated compared to non-treated plants in King Kong2 at 2 dpi in midstems and in all organs at 5 dpi, and in Hawaii 7998 (resistant) in all organs at 2 dpi. Differences between genotypes were obvious on midstem level (5 dpi), where bacterial populations were generally significantly lower compared to roots. Increased tolerance was observed in genotypes L390 and King Kong2 with silicon treatment. Silicon accumulated in roots and was low in stems and leaves. Inoculation with Ralstonia solanacearum did not significantly affect silicon uptake and distribution. Negative correlations between root silicon content and bacterial numbers of midstems in genotypes Hawaii 7998 and King Kong2 suggested an induced resistance. Indications for an influence of host genotype and silicon treatment on the phenotypic conversion of R. solanacearum strain To-udk2-sb from fluidal to non-fluidal colonies in planta were observed. This is the first report on the effect of silicon on a bacterial disease and in a silicon-non-accumulator plant.
Abstract: Results obtained by flow cytometry allow conclusions to be drawn about how the physiological states of Ralstonia eutropha JMP134 are connected with survival strategies under distinct growth conditions. During both feast and famine conditions the cells were found to proceed through sharply separated phases of life. Two sources of carbon and energy, one poor (0.02% phenol) and one rich (0.2% pyruvate and 0.1% yeast extract) were chosen to study the cellular responses. Despite the major differences in carbon source, when growth stages of the bacteria on the two substrates were characterised in batch growth, only minor differences were found in the time course of the membrane potential related fluorescence intensity (MPRFI). This also applied to the rRNA content and the size-correlated forward scatter (FSC) signal of the cells, both of which increased to high levels during the (early) exponential growth phase. On the rich medium, DNA synthesis initially occurred in an uncoupled manner, then a high rate of PHB formation followed when nutrients began to be limiting. Under famine conditions, the cellular responses were much more complex. PHB was synthesised, then DNA synthesis occurred in a eukaryotic mode, to be succeeded by renewed PHB synthesis. To obtain defined cell physiological states, the chemostat technique was used in addition to batch experiments. The results obtained clearly indicated that key events in cell physiology, including initiation of DNA replication and overflow metabolism, occurred in a hierarchically ordered manner and were tightly correlated with changes in the environmental conditions of the bacterial cells.
Abstract: Self-assembling peptides have emerged as an attractive scaffold material for tissue engineering, yet the expense associated with solid phase chemical synthesis has limited their broad use. In addition, the fidelity of chemical synthesis constrains the length of polypeptides that can be produced homogeneously by this method. Template-derived biosynthesis by recombinant DNA technology may overcome both of these problems. However, recovery of polypeptides from recombinant protein expression systems typically involves multi-step purification schemes. In this study, we report an integrated approach to recombinantly produce and purify self-assembling peptides from the recently developed expression host Ralstonia eutropha. The purification is based on the specific affinity of carbohydrate binding modules (CBMs) to cellulose. In a first step, we identified CBMs that express well in R. eutropha by assembling a fusion library of green fluorescent protein (GFP) and CBMs and determining the fluorescence of cell-free extracts. Three GFP::CBM fusions were found to express at levels similar to GFP alone, of which two CBMs were able to mediate cellulose binding of the GFP::CBM fusion. These two CBMs were then fused to multiple repeats of the self-assembling peptide RAD16-I::E (n-RADARADARADARADAE-c). The fusion protein CBM::E::(RAD16-I::E)4 was expressed in R. eutropha and purified using the CBM’s affinity for cellulose. Subsequent proteolytic cleavage with endoproteinase GluC liberated RAD16-I::E peptide monomers with similar properties to the chemically synthesized counterpart RAD16-I.
Abstract:Ralstonia solanacearum causes bacterial wilt, a soil-borne vascular disease of many crop plants. Plant cell wall-degrading polygalacturonases (PGs) are significant virulence factors for this pathogen. R. solanacearum mutants lacking PehA (an endo-PG), or PehB (an exo-PG), or both PehA and PehB are all less virulent than the wild-type strain on wounded eggplants. To more closely mimic the natural infection process, unwounded tomato plants were soil-soak inoculated with wild-type and PG mutant strains. All three PG mutants were significantly reduced in virulence on intact tomato plants, despite surviving well in potting mix. Over the course of disease development, populations of PG mutant and wild-type bacteria in plants were significantly different. The PG mutants, especially the two pehA mutants, colonized fewer tomato stems, colonized stems more slowly, and had lower mean bacterial populations in stems than the wild-type strain. These results suggest that PehA and PehB are necessary for rapid host colonization, and that production of these enzymes contributes quantitatively to the ability of R. solanacearum to colonize host vascular tissue and wilt plants. A threshold bacterial population of around 1 × 108 cfu in the centimetre of tomato stem directly above the cotyledons correlated with the appearance of wilt symptoms.
Abstract: The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (103–108 cells ml-1) to amplification of DNA
showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 * 107 cells g-1 of soil in the mycorrhizosphere and 7.0 * 106 cells g-1 in the bulk soil. We evaluated the W. basilensis3 cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 102 to 107 cells g-1 soil (104 –109 cells ml-1). Cell densities of W. basilensis of >106 cells g-1 of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.
Abstract: The mature lipase LipA and its 56aa-truncated chaperone LipBhis (with 6£his-tag) from Ralstonia sp. M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively. The simply purified lipase LipA was effectively refolded by Ni–NTA purified chaperone LipBhis in molar ratio 1:1 at 4°C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50–55°C and was stable up to 45°C with more than 84% activity retention. The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0–10.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137–334%), but inhibited by 1-butanol and acetonitrile (40–86%). Metal ions Cu2+, Sn2+, Mn2+, Mg2+, and Ca2+ stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn2+ significantly inhibited the enzyme with the residual activity of 30–65% and Fe3+ to a lesser degree (activity retention of 77–86%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222–330%) and 0.2–1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52%. However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31–89%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1–10mM) inhibited moderately the lipase with remaining activity of 57–105%. The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6). In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase. The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222%), and wheatgerm oil (210%) in comparison to olive oil (100%).
Abstract: Three diagrammatic grading keys were designed for the assessment of the severity of late blight (caused by Phytophthora infestans) in tomato leaves. Simplified and broad keys considered, respectively, six (3, 12, 22, 40, 60 and 77%) and eight (3, 6, 12, 22, 40, 60, 77 and 90%) levels of disease severity, whilst a modified key based on a previous proposal for potato late blight considered six levels (1, 5, 10, 16, 32 and 50%). The keys were validated by 24 evaluators who assessed digital images of tomato leaves exhibiting different areas with lesions. Evaluator errors were compared using a mixed model in which evaluators were considered as random effects and the keys and evaluations as fixed effects. The accuracy and precision of the evaluators were compared by simple linear regression between the estimated and actual values of disease severity. The repeatability of evaluators was assessed using Pearson's correlation coefficient. There was significant (P ≤ 0·001) variability amongst the errors made by evaluators, although the precision of each of the three keys was high with a coefficient of determination (R2) of 0·96, 0·93 and 0·83 for the simplified, broad and modified key, respectively. Repeatability of estimations amongst the evaluators was adequate (correlation coefficients of 0·91, 0·91 and 0·90 for the three keys, respectively). The simplified and broad keys resulted in higher precision and accuracy for the estimation of severity than did the modified key. Since the simplified key considers a smaller number of disease severity levels, its use is recommended in the assessment of late blight in tomato leaves.
Abstract: Data on plant pathogenic oomycetes are scarce and little is known about the early events leading to the onset of infection. The aim of this work was to analyze the penetration process of the soil-borne plant pathogen Phytophthora parasitica, which has a wide host range. Here, we performed a cytological analysis of the colonization of the first plant cell and developed an inoculation assay for characterizing the entire penetration process through cellular and molecular analyses. We showed that P. parasitica infects roots by producing a specialized structure, the appressorium. We produced the first cDNA library for the penetrating stage of a Phytophthora species and showed it to be highly enriched in pathogenicity-related sequences. These included coding sequences for many cell-degrading enzymes, effectors such as RXLR-containing proteins and proteins involved in protection against plant defense responses.
Characterization of the appressorium cDNA library and identification of genes overrepresented early in P. parasitica infection provided us with an unprecedented opportunity to decipher the molecular mechanisms involved in penetration of the plant cells during the initiation of infection by a soil-borne oomycete.
Abstract: The double setting of two seed tubers is introduced as a new method for research on primary stem blight of potato, Solanum tuberosum L. caused by Phytophthora infestans (Mont.) de Bary). The principal focus of this experimental layout is the planting of a healthy seed tuber adjacent to an inoculated one, termed double setting. The infected tuber serves as a source of inoculum, enabling the disease to spread to the neighboring plant, where stem infections occur. The new method was compared to the common procedure of direct inoculation of tubers. The rate of emergence using double setting was significantly higher compared to direct inoculation. Additionally, stem blight was more intense and generally occurred earlier. Direct inoculation proved to be less suitable for research where the occurrence of higher percentages of stem blight symptoms is necessary. Further more, the double setting method generally permits insight into the spread of the disease from latent infected tubers to healthy ones under the natural conditions within the potato hill.
Abstract: Species distribution models (SDMs) based on statistical relationships between occurrence data and underlying environmental conditions are increasingly used to predict spatial patterns of biological invasions and prioritize locations for early detection and control of invasion outbreaks. However, invasive species distribution models (iSDMs) face special challenges because (i) they typically violate SDM's assumption that the organism is in equilibrium with its environment, and (ii) species absence data are often unavailable or believed to be too difficult to interpret. This often leads researchers to generate pseudo-absences for model training or utilize presence-only methods, and to confuse the distinction between predictions of potential vs. actual distribution. We examined the hypothesis that true-absence data, when accompanied by dispersal constraints, improve prediction accuracy and ecological understanding of iSDMs that aim to predict the actual distribution of biological invasions. We evaluated the impact of presence-only, true-absence and pseudo-absence data on model accuracy using an extensive dataset on the distribution of the invasive forest pathogen Phytophthora ramorum in California. Two traditional presence/absence models (generalized linear model and classification trees) and two alternative presence-only models (ecological niche factor analysis and maximum entropy) were developed based on 890 field plots of pathogen occurrence and several climatic, topographic, host vegetation and dispersal variables. The effects of all three possible types of occurrence data on model performance were evaluated with receiver operating characteristic (ROC) and omission/commission error rates. Results show that prediction of actual distribution was less accurate when we ignored true-absences and dispersal constraints. Presence-only models and models without dispersal information tended to over-predict the actual range of invasions. Models based on pseudo-absence data exhibited similar accuracies as presence-only models but produced spatially less feasible predictions. We suggest that true-absence data are a critical ingredient not only for accurate calibration but also for ecologically meaningful assessment of iSDMs that focus on predictions of actual distributions.
Abstract