scholarly journals Bioluminescent Xanthomonas hortorum pv. gardneri as a Tool to Quantify Bacteria in Planta, Screen Germplasm, and Identify Infection Routes on Leaf Surfaces

2021 ◽  
Vol 12 ◽  
Author(s):  
Eduardo Bernal ◽  
Loïc Deblais ◽  
Gireesh Rajashekara ◽  
David M. Francis
Keyword(s):  
Electron Microscopy ◽  
Disease Severity ◽  
Bioluminescence Imaging ◽  
Field Trials ◽  
In Planta ◽  
Bacterial Populations ◽  
Inoculation Techniques ◽  
Leaf Surfaces ◽  
Infection Processes ◽  
Susceptible Tomato

Imaging technology can provide insight into biological processes governing plant-pathogen interactions. We created and used a bioluminescent strain of Xanthomonas hortorum pv. gardneri (Xgb) to quantify infection processes in plants using tomato as a model. An X. hortorum pv. gardneri is one of the four Xanthomonas species that causes bacterial spots in tomatoes. We used Xgb to quantify bacterial growth in planta, to assess disease severity in resistant and susceptible tomato lines, and to observe infection routes in leaves. A positive and significant linear correlation r (67) = 0.57, p ≤ 0.0001 was observed between bioluminescence signals emitted by Xgb in planta and bacterial populations determined through dilution plating. Based on bioluminescence imaging, resistant and susceptible tomato lines had significantly different average radiances. In addition, there was a positive and significant correlation r = 0.45, p = 0.024 between X. hortorum pv. gardneri-inoculated tomato lines evaluated by bioluminescence imaging and tomatoes rated in the field using the Horsfall-Barrat Scale. Heritability was calculated to compare the genetic variance for disease severity using bioluminescence imaging and classical field ratings. The genetic variances were 25 and 63% for bioluminescence imaging and field ratings, respectively. The disadvantage of lower heritability attained by bioluminescence imaging may be offset by the ability to complete germplasm evaluation experiments within 30 days rather than 90–120 days in field trials. We further explored X. hortorum pv. gardneri infection routes on leaves using spray and dip inoculation techniques. Patterns of bioluminescence demonstrated that the inoculation technique affected the distribution of bacteria, an observation verified using scanning electron microscopy (SEM). We found significant non-random distributions of X. hortorum pv. gardneri on leaf surfaces with the method of inoculation affecting bacterial distribution on leaf surfaces at 4 h postinoculation (hpi). At 18 hpi, regardless of inoculation method, X. hortorum pv. gardneri localized on leaf edges near hydathodes based on bioluminescence imaging and confirmed by electron microscopy. These findings demonstrated the utility of bioluminescent X. hortorum pv. gardneri to estimate bacterial populations in planta, to select for resistant germplasm, and to detect likely points of infection.

scholarly journals Zinc phosphate protects tomato plants against Pseudomonas syringae pv. tomato

2021 ◽  
Author(s):  
Mara Quaglia ◽  
Marika Bocchini ◽  
Benedetta Orfei ◽  
Roberto D’Amato ◽  
Franco Famiani ◽  
...  
Keyword(s):  
Disease Severity ◽  
Pseudomonas Syringae ◽  
Zinc Phosphate ◽  
Plant Defence ◽  
In Planta ◽  
Tomato Plants ◽  
Pathogenesis Related Protein ◽  
Defence Responses ◽  
Plant Defence Responses

AbstractThe purpose of this study was to determine whether zinc phosphate treatments of tomato plants (Solanum lycopersicum L.) can attenuate bacterial speck disease severity through reduction of Pseudomonas syringae pv. tomato (Pst) growth in planta and induce morphological and biochemical plant defence responses. Tomato plants were treated with 10 ppm (25.90 µM) zinc phosphate and then spray inoculated with strain DAPP-PG 215, race 0 of Pst. Disease symptoms were recorded as chlorosis and/or necrosis per leaf (%) and as numbers of necrotic spots. Soil treatments with zinc phosphate protected susceptible tomato plants against Pst, with reductions in both disease severity and pathogen growth in planta. The reduction of Pst growth in planta combined with significantly higher zinc levels in zinc-phosphate-treated plants indicated direct antimicrobial toxicity of this microelement, as also confirmed by in vitro assays. Morphological (i.e. callose apposition) and biochemical (i.e., expression of salicylic-acid-dependent pathogenesis-related protein PR1b1 gene) defence responses were induced by the zinc phosphate treatment, as demonstrated by histochemical and qPCR analyses, respectively. In conclusion, soil treatments with zinc phosphate can protect tomato plants against Pst attacks through direct antimicrobial activity and induction of morphological and biochemical plant defence responses.

scholarly journals Spatial-Temporal and Quantitative Analysis of Growth and EPS I Production by Ralstonia solanacearum in Resistant and Susceptible Tomato Cultivars

1999 ◽  
Vol 89 (12) ◽  
pp. 1233-1239 ◽  
Author(s):  
J. A. McGarvey ◽  
T. P. Denny ◽  
M. A. Schell
Keyword(s):  
Ralstonia Solanacearum ◽  
Antimicrobial Activities ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vascular Bundles ◽  
Susceptible Cultivar ◽  
Bacterial Populations ◽  
Resistant Cultivars ◽  
Cell Densities ◽  
Almost All ◽  
Susceptible Tomato

One susceptible and two resistant cultivars of tomato were tested for differences in infection by Ralstonia solanacearum and for the subsequent multiplication, colonization, and production of the wilt-inducing virulence factor, exopolysaccharide I (EPS I). Bacterial ingress into the taproot was fastest in the susceptible cv. Marion, followed by the resistant cvs. L285 (fivefold slower) and Hawaii 7996 (15-fold slower). Once inside the taproot, R. solanacearum colonized, to some extent, almost all regions of the resistant and susceptible plants. However, colonization occurred sooner in the susceptible than in the resistant cultivars, as measured by viablecell counts of bacteria in the midstems. Rates of multiplication and maximum bacterial cell densities were also greater in the susceptible than in the resistant cultivars. Growth experiments utilizing xylem fluid from infected and uninfected plants indicated that neither antimicrobial activities nor reduced levels of growth-supporting nutrients in the xylem fluids were responsible for the reduced bacterial multiplication in the resistant cultivars. Quantification of EPS I in the infected plants, using an enzyme-linked immunosorbent assay, revealed that the bacterial populations in the susceptible cultivar produced greater amounts of EPS I per plant than those in the resistant cultivars. Immunofluorescence microscopy using antibodies against either EPS I or R. solanacearum cells revealed that bacteria and EPS I were distributed throughout the vascular bundles and intercellular spaces of the pith in the susceptible cultivar, whereas in the resistant cultivars, bacteria and EPS I were restricted to the vascular tissues.

scholarly journals Epidemiology and management of bacterial leaf spot on watermelon caused byPseudomonas syringae

Plant Disease ◽  
2017 ◽  
Vol 101 (7) ◽  
pp. 1222-1229 ◽  
Author(s):  
E. A. Newberry ◽  
L. Ritchie ◽  
B. Babu ◽  
T. Sanchez ◽  
K. A. Beckham ◽  
...  
Keyword(s):  
Disease Severity ◽  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Negative Relationship ◽  
Disease Outbreaks ◽  
Field Trials ◽  
Multiple Regression Model ◽  
Bacterial Leaf Spot ◽  
Progress Curve ◽  
Cool Conditions

Bacterial leaf spot of watermelon caused by Pseudomonas syringae has been an emerging disease in the southeastern United States in recent years. Disease outbreaks in Florida were widespread from 2013 to 2014 and resulted in foliar blighting at the early stages of the crop and transplant losses. We conducted a series of field trials at two locations over the course of two years to examine the chemical control options that may be effective in management of this disease, and to investigate the environmental conditions conducive for bacterial leaf spot development. Weekly applications of acibenzolar-S-methyl (ASM) foliar, ASM drip, or copper hydroxide mixed with ethylene bis-dithiocarbamate were effective in reducing the standardized area under the disease progress curve (P < 0.05). Pearson’s correlation test demonstrated a negative relationship between the average weekly temperature and disease severity (–0.77, P = 0.0002). When incorporated into a multiple regression model with the square root transformed average weekly rainfall, these two variables accounted for 71% of the variability observed in the weekly disease severity (P < 0.0001). This information should be considered when choosing the planting date for watermelon seedlings as the cool conditions often encountered early in the spring season are conducive for bacterial leaf spot development.

THE ELECTRON MICROSCOPY OF LEAF SURFACES PRESERVED IN PEAT

1966 ◽  
Vol 44 (4) ◽  
pp. 421-427 ◽  
Author(s):  
John M. Stewart ◽  
Edward A. C. Follett
Keyword(s):  
Electron Microscopy ◽  
Cell Wall ◽  
Calluna Vulgaris ◽  
Surface Features ◽  
Peat Deposits ◽  
Leaf Surfaces ◽  
Definition Of ◽  
Peat Formation ◽  
Physical And Chemical ◽  
Cuticular Surface

Phragmites communis, Eriophorum vaginatum, Calluna vulgaris, and Sphagnum palustre are representative of plants whose remains are frequently encountered in Scottish peat deposits. The effects of preservation in peat on the surface features of their leaves were followed by electron microscopy. Wax projections were observed on the surfaces of mature living leaves of Phragmites and Eriophorum but not on Calluna or Sphagnum. Details of cell wall outlines and stomata (or pores) were clearly defined in Phragmites, Eriophorum, and Sphagnum, but obscured in Calluna. The previous year's leaves differed by displaying a general absence of wax projections, an erosion of the cuticular surface, which took the form of either a loss in definition of the cell wall outlines or a definite etching of the surface, and the presence of numerous microorganisms. The surface features of preserved leaves exhibited to a greater degree this erosion of cell wall outline and cuticular surface. This preliminary study has indicated that major alterations in the submicroscopic features of cuticularized leaf surfaces occur at the leaf litter stage. The primary agents responsible for this degradation would appear to be microorganisms in conjunction with the physical and chemical processes of peat formation.

scholarly journals Considerations for Verticillium Wilt Resistance Evaluation in Potato

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 360-367 ◽  
Author(s):  
K. E. Frost ◽  
D. I. Rouse ◽  
S. H. Jansky
Keyword(s):  
Disease Severity ◽  
Verticillium Wilt ◽  
Yield Loss ◽  
Control Culture ◽  
In Planta ◽  
Disease Symptoms ◽  
Accuracy And Precision ◽  
Population Sizes ◽  
Pathogen Colonization ◽  
Time Accuracy

Verticillium wilt (Vw), caused by the soilborne fungi Verticillium dahliae and V. albo-atrum, is an important disease of potato (Solanum tuberosum). Host plant resistance is a promising method of Vw control. Culture-based methods that quantify the pathogen in host tissue often are used for Vw resistance screening. To evaluate the processing time, accuracy, and precision of these methods, 46 clones were planted in a field naturally infested with V. dahliae to collect data on visual disease symptoms, pathogen colonization, and yield. In 2002, disease severity explained 4.34% of the variability of yield loss, but the linear relationship between stem colonization and yield loss was not significant. In 2003, stem colonization explained 57.5% of the variability of yield loss, whereas disease severity explained 1.7% of the variability of yield loss. Correlations comparing clone ranks from repeated pathogen measurements indicated that culturing sap from individual stems or bulked stems generated more repeatable clone rankings than culturing dried stems. Clone rankings were more repeatable between years if pathogen measurements were made earlier in the growing season. The results indicate a need to characterize the effect of the environment on the relationship among pathogen population sizes in planta, disease symptoms, and yield loss.

scholarly journals Efficacy of Variable Tetraconazole Rates Against Cercospora beticola Isolates with Differing In Vitro Sensitivities to DMI Fungicides

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1749-1756 ◽  
Author(s):  
Melvin D. Bolton ◽  
Viviana Rivera-Varas ◽  
Luis E. del Río Mendoza ◽  
Mohamed F. R. Khan ◽  
Gary A. Secor
Keyword(s):  
Sugar Beet ◽  
Disease Severity ◽  
Cercospora Beticola ◽  
In Planta ◽  
Sensitivity Testing ◽  
In Vitro Sensitivity ◽  
Wide Range ◽  
Fungicide Efficacy ◽  
Dmi Fungicides

Cercospora leaf spot (CLS) of sugar beet is caused by the fungus Cercospora beticola. CLS management practices include the application of the sterol demethylation inhibitor (DMI) fungicides tetraconazole, difenoconazole, and prothioconazole. Evaluating resistance to DMIs is a major focus for CLS fungicide resistance management. Isolates were collected in 1997 and 1998 (baseline sensitivity to tetraconazole, prothioconazole, or difenoconazole) and 2007 through 2010 from the major sugar-beet-growing regions of Minnesota and North Dakota and assessed for in vitro sensitivity to two or three DMI fungicides. Most (47%) isolates collected in 1997–98 exhibited 50% effective concentration (EC50) values for tetraconazole of <0.01 μg ml–1, whereas no isolates could be found in this EC50 range in 2010. Since 2007, annual median and mean tetraconazole EC50 values have generally been increasing, and the frequency of isolates with EC50 values >0.11 μg ml–1 increased from 2008 to 2010. In contrast, the frequency of isolates with EC50 values for prothioconazole of >1.0 μg ml–1 has been decreasing since 2007. Annual median difenoconazole EC50 values appears to be stable, although annual mean EC50 values generally have been increasing for this fungicide. Although EC50 values are important for gauging fungicide sensitivity trends, a rigorous comparison of the relationship between in vitro EC50 values and loss of fungicide efficacy in planta has not been conducted for C. beticola. To explore this, 12 isolates exhibiting a wide range of tetraconazole EC50 values were inoculated to sugar beet but no tetraconazole was applied. No relationship was found between isolate EC50 value and disease severity. To assess whether EC50 values are related to fungicide efficacy in planta, sugar beet plants were sprayed with various dilutions of Eminent, the commercial formulation of tetraconazole, and subsequently inoculated with isolates that exhibited very low, medium, or high tetraconazole EC50 values. The high EC50 isolate caused significantly more disease than isolates with medium or very low EC50 values at the field application rate and most reduced rates. Because in vitro sensitivity testing is typically carried out with the active ingredient of the commercial fungicide, we investigated whether loss of disease control was the same for tetraconazole as for the commercial product Eminent. The high EC50 isolate caused more disease on plants treated with tetraconazole than Eminent but disease severity was not different between plants inoculated with the very low EC50 isolate.

Bacterial colonization patterns of intactPinus sylvestrismycorrhizospheres in dry pine forest soil: an electron microscopy study

1997 ◽  
Vol 43 (11) ◽  
pp. 1017-1035 ◽  
Author(s):  
E. -L. Nurmiaho-Lassila ◽  
S. Timonen ◽  
K. Haahtela ◽  
R. Sen
Keyword(s):  
Electron Microscopy ◽  
Pinus Sylvestris ◽  
Forest Soil ◽  
Bacterial Colonization ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Paxillus Involutus ◽  
Intracellular Bacteria ◽  
Bacterial Populations ◽  
Suillus Bovinus

The bacterial populations associated with different plant and fungal habitats of intact Pinus sylvestris – Suillus bovinus or Pinus sylvestris – Paxillus involutus ectomycorrhizospheres grown in natural forest soil were examined by scanning and transmission electron microscopy. Surfaces of nonmycorrhizal Pinus sylvestris roots hosted large numbers of morphologically distinct bacteria. Bacteria were detected on the mantle surfaces and at inter- and intra-cellular locations in the mantle and Hartig net of Suillus bovinus mycorrhizas. The fungal strands were colonized by only a few bacteria unlike the outermost external fine hyphae on which extensive monolayers of bacteria were attached. The mycorrhizas of Paxillus involutus were mostly devoid of bacteria, but the intact external mycelium supported both bacterial colonies and solitary bacteria. Intracellular bacteria were not present in Paxillus involutus hyphae. In both mycorrhizal systems, bacterial aggregation and attachment to hyphae were mediated with electron-dense or -translucent material. Our study shows that the Pinus sylvestris mycorrhizospheres formed by two different ectomycorrhizal fungi are clearly dissimilar habitats for mycorrhizosphere-associated bacteria. Additionally, the spatially and physiologically defined mycorrhizosphere habitats were shown to host distinct populations of bacteria.Key words: ectomycorrhiza, intracellular bacteria, Paxillus involutus, soil bacteria, Suillus bovinus.

scholarly journals Subgroup Characteristics of Marine Methane-Oxidizing ANME-2 Archaea and Their Syntrophic Partners as Revealed by Integrated Multimodal Analytical Microscopy

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Shawn E. McGlynn ◽  
Grayson L. Chadwick ◽  
Ariel O'Neill ◽  
Mason Mackey ◽  
Andrea Thor ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Methane Oxidation ◽  
Three Dimensional ◽  
Fluorescence Labeling ◽  
Anaerobic Oxidation Of Methane ◽  
Methane Seep ◽  
Bacterial Populations ◽  
Symbiotic Associations ◽  
Iron Mineral ◽  
Oxidation Of Methane

ABSTRACTPhylogenetically diverse environmental ANME archaea and sulfate-reducing bacteria cooperatively catalyze the anaerobic oxidation of methane oxidation (AOM) in multicelled consortia within methane seep environments. To better understand these cells and their symbiotic associations, we applied a suite of electron microscopy approaches, including correlative fluorescencein situhybridization-electron microscopy (FISH-EM), transmission electron microscopy (TEM), and serial block face scanning electron microscopy (SBEM) three-dimensional (3D) reconstructions. FISH-EM of methane seep-derived consortia revealed phylogenetic variability in terms of cell morphology, ultrastructure, and storage granules. Representatives of the ANME-2b clade, but not other ANME-2 groups, contained polyphosphate-like granules, while some bacteria associated with ANME-2a/2c contained two distinct phases of iron mineral chains resembling magnetosomes. 3D segmentation of two ANME-2 consortium types revealed cellular volumes of ANME and their symbiotic partners that were larger than previous estimates based on light microscopy. Polyphosphate-like granule-containing ANME (tentatively termed ANME-2b) were larger than both ANME with no granules and partner bacteria. This cell type was observed with up to 4 granules per cell, and the volume of the cell was larger in proportion to the number of granules inside it, but the percentage of the cell occupied by these granules did not vary with granule number. These results illuminate distinctions between ANME-2 archaeal lineages and partnering bacterial populations that are apparently unified in their ability to perform anaerobic methane oxidation.IMPORTANCEMethane oxidation in anaerobic environments can be accomplished by a number of archaeal groups, some of which live in syntrophic relationships with bacteria in structured consortia. Little is known of the distinguishing characteristics of these groups. Here, we applied imaging approaches to better understand the properties of these cells. We found unexpected morphological, structural, and volume variability of these uncultured groups by correlating fluorescence labeling of cells with electron microscopy observables.

scholarly journals In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

2009 ◽  
Vol 76 (1) ◽  
pp. 264-274 ◽  
Author(s):  
M.-L. Foucault ◽  
L. Thomas ◽  
S. Goussard ◽  
B. R. Branchini ◽  
C. Grillot-Courvalin
Keyword(s):  
Escherichia Coli ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Direct Method ◽  
Firefly Luciferase ◽  
Intestinal Colonization ◽  
Bacterial Populations ◽  
Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

Dynamics of RP4 plasmid transfer between Xanthomonas campestris pv. corylina and Erwinia herbicola in hazelnut tissues, in planta

1986 ◽  
Vol 32 (11) ◽  
pp. 835-841 ◽  
Author(s):  
Charles Manceau ◽  
Louis Gardan ◽  
Martine Devaux
Keyword(s):  
Pseudomonas Fluorescens ◽  
Xanthomonas Campestris ◽  
Plasmid Transfer ◽  
In Planta ◽  
Erwinia Herbicola ◽  
Bacterial Populations ◽  
Physiological Conditions ◽  
Factor Governing ◽  
Host Tissues ◽  
Main Factor

We have shown that the transfer of plasmid RP4 took place between two strains of Xanthomonas campestris pv. corylina and from Xanthomonas campestris pv. corylina to Erwinia herbicola and vice versa when the strains were inoculated in hazelnut tissues in an experimental orchard. Transfer occurred throughout all seasons and was independent of the physiological conditions of the host tissues. The main factor governing transfer in planta appeared to be the frequency of contacts among donors and recipients. The frequency of transfer was correlated with the level of bacterial populations. RP4 was stable in the inoculated strains of Xanthomonas campestris pv. corylina as well as in Erwinia herbicola in hazelnut tissue. RP4 was detected in two isolates of resident epiphytic microflora, Pseudomonas fluorescens and Erwinia herbicola.

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