Further Characterization of Genetically Distinct Groups of Acidovorax citrulli Strains

Bacterial fruit blotch of cucurbits (BFB) is caused by the gram-negative bacterium Acidovorax citrulli, whose populations can be distinguished into two genetically distinct groups, I and II. Based on visual assessment of BFB severity on cucurbit seedlings and fruit after inoculation under greenhouse conditions, group I A. citrulli strains have been reported to be moderately to highly virulent on several cucurbit hosts, whereas group II strains have exhibited high virulence on watermelon but low virulence on other cucurbits. Additionally, group I strains are recovered from a range of cucurbit hosts, while group II strains are predominantly found on watermelon. The goal of this research was to develop tools to characterize and rapidly distinguish group I and II A. citrulli strains. We first sought to determine whether quantification of A. citrulli colonization of cucurbit seedling tissue reflects the differences between group I and II strains established by visual assessment of BFB symptom severity. Spray inoculation of melon seedlings with cell suspensions containing approximately 1 × 104 CFU/ml resulted in significantly higher (P = 0.01) population growth of M6 (group I; mean area under population growth curve [AUPGC] = 43.73) than that of AAC00-1 (group II; mean AUPGC = 39.33) by 10 days after inoculation. We also investigated the natural spread of bacterial cells and the resulting BFB incidence on watermelon and melon seedlings exposed to three group I and three group II A. citrulli strains under mist chamber conditions. After 5 days of exposure, the mean BFB incidence on melon seedlings exposed to representative group II A. citrulli strains was significantly lower (25 and 3.98% in experiments 1 and 2, respectively) than on melon seedlings exposed to representative group I strains (94.44 and 76.11% in experiments 1 and 2, respectively), and on watermelon seedlings exposed to representative group I and II strains (70 to 93.33%). Finally, we developed a polymerase chain reaction assay based on the putative type III secretion effector gene, Aave_2166, to rapidly distinguish group I and II A. citrulli strains. This assay will be important for future epidemiological studies on BFB.

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Genetically Distinct Acidovorax citrulli Strains Display Cucurbit Fruit Preference Under Field Conditions

Phytopathology ◽

10.1094/phyto-10-19-0389-r ◽

2020 ◽

Vol 110 (5) ◽

pp. 973-980 ◽

Cited By ~ 1

Author(s):

Mei Zhao ◽

Bhabesh Dutta ◽

Xuelin Luo ◽

Saul Burdman ◽

Ron Walcott

Keyword(s):

Field Conditions ◽

Distribution Data ◽

Specific Pcr ◽

Acidovorax Citrulli ◽

Group I ◽

Fruit Preference ◽

Direct Data ◽

Group Ii ◽

Specific Pcr Assay ◽

Field Plots

Strains of Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, can be assigned to two groups, I and II. The natural association of group I and II strains with different cucurbit species suggests host preference; however, there are no direct data to support this hypothesis under field conditions. Hence, the objective of this study was to assess differences in the prevalence of group I and II A. citrulli strains on cucurbit species in the field. From 2017 to 2019, we used group I and II strains to initiate BFB outbreaks in field plots planted with four cucurbit species. At different times, we collected symptomatic tissues and assayed them for group I and II strains using a group-specific PCR assay. Binary distribution data analysis revealed that the odds of melon, pumpkin, and squash foliage infection by group I strains were 21.7, 11.5, and 22.1 times greater, respectively, than the odds of watermelon foliage infection by the group I strain (P < 0.0001). More strikingly, the odds of melon fruit infection by the group I strain were 97.5 times greater than watermelon fruit infection by the same strain (P < 0.0001). Unexpectedly, some of the group II isolates recovered from the 2017 and 2019 studies were different from the group II strains used as inocula. Overall, data from these experiments confirm that A. citrulli strains exhibit a preference for watermelon and melon, which is more pronounced in fruit tissues.

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Further Evidence of Cucurbit Host Specificity among Acidovorax citrulli Groups Based on a Detached Melon Fruit Pathogenicity Assay

Phytopathology ◽

10.1094/phyto-11-16-0416-r ◽

2017 ◽

Vol 107 (11) ◽

pp. 1305-1311 ◽

Cited By ~ 9

Author(s):

Lichun Yan ◽

Baishi Hu ◽

Gong Chen ◽

Mei Zhao ◽

Ron R. Walcott

Keyword(s):

Host Specificity ◽

Crop Production ◽

Host Preference ◽

Specific Capacity ◽

Effector Proteins ◽

Acidovorax Citrulli ◽

Group I ◽

Group Ii ◽

Melon Fruit ◽

Fruit Rind

Bacterial fruit blotch, caused by the gram-negative bacterium Acidovorax citrulli, is a serious economic threat to cucurbit crop production worldwide. A. citrulli strains can be divided into two genetically distinct groups, with group I strains infecting a range of cucurbit species and group II strains being predominantly associated with watermelon. Group I and II A. citrulli strains differ in their arsenal of type III secreted (T3S) effector proteins and we hypothesize that these effectors are critical for cucurbit host preference. However, the pathogenicity or virulence assays used for A. citrulli, including infiltration of seedling cotyledons and mature fruit rind tissues with cell suspensions and spray inoculation of seedlings, lack the sensitivity to consistently distinguish strains of the two groups. Here, we describe an immature, detached melon fruit assay based on ‘Joaquin Gold’ melon (Syngenta, Rogers Brand) that clearly indicates differences in host specificity between group I and II A. citrulli strains. Using this assay, four group I strains (M6, AAC213-52, AAC213-55, and XJL12) induced typical water-soaked lesions in melon fruit rind tissue 7 to 10 days after pinprick inoculation. In contrast, four group II strains (AAC00-1, AAC213-44, AAC213-47, and AAC213-48) did not induce water-soaked lesions on detached melon fruit rinds during the same period. These data suggest that group I A. citrulli strains have a specific capacity to infect immature Joaquin Gold melon fruit, whereas group II strains do not. Interestingly, this differential pathogenicity phenotype was not observed on foliar seedling tissues of the same melon cultivar, suggesting that host preference of A. citrulli strains is specific to immature fruit tissues. Using the immature melon fruit inoculation assay, a T3S system mutant of the group I A. citrulli strain, M6 (M6ΔhrcV), failed to induce water soaking. This indicates that T3S effectors are involved in A. citrulli cucurbit host preference, and that this assay is suitable for future studies of unique T3S effectors that distinguish group I and II strains.

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Strains of the Group I Lineage of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch of Cucurbitaceous Crops, are Predominant in Brazil

Phytopathology ◽

10.1094/phyto-05-16-0205-r ◽

2016 ◽

Vol 106 (12) ◽

pp. 1486-1494 ◽

Cited By ~ 9

Author(s):

Gustavo M. Silva ◽

Ricardo M. Souza ◽

Lichun Yan ◽

Rui S. Júnior ◽

Flavio H. V. Medeiros ◽

...

Keyword(s):

Genetic Diversity ◽

Population Structure ◽

Effect Of Temperature ◽

Pfge Analysis ◽

Bacterial Fruit Blotch ◽

Acidovorax Citrulli ◽

Group I ◽

Pathogenicity Tests ◽

Group Ii

Bacterial fruit blotch (BFB), caused by the seedborne bacterium Acidovorax citrulli, is an economically important threat to cucurbitaceous crops worldwide. Since the first report of BFB in Brazil in 1990, outbreaks have occurred sporadically on watermelon and, more frequently, on melon, resulting in significant yield losses. At present, the genetic diversity and the population structure of A. citrulli strains in Brazil remain unclear. A collection of 74 A. citrulli strains isolated from naturally infected tissues of different cucurbit hosts in Brazil between 2000 and 2014 and 18 A. citrulli reference strains from other countries were compared by pulsed-field gel electrophoresis (PFGE), multilocus sequence analysis (MLSA) of housekeeping and virulence-associated genes, and pathogenicity tests on seedlings of different cucurbit species. The Brazilian population comprised predominantly group I strains (98%), regardless of the year of isolation, geographical region, or host. Whole-genome restriction digestion and PFGE analysis revealed that three unique and previously unreported A. citrulli haplotypes (assigned as haplotypes B22, B23, and B24) occurred in Brazil. The greatest diversity of A. citrulli (four haplotypes) was found among strains collected from the northeastern region of Brazil, which accounts for more than 90% of the country’s melon production. MLSA clearly distinguished A. citrulli strains into two well-supported clades, in agreement with observations based on PFGE analysis. Five Brazilian A. citrulli strains, representing different group I haplotypes, were moderately aggressive on watermelon seedlings compared with four group II strains that were highly aggressive. In contrast, no significant differences in BFB severity were observed between group I and II A. citrulli strains on melon and squash seedlings. Finally, we observed a differential effect of temperature on in vitro growth of representative group I and II A. citrulli haplotypes. Specifically, of 18 group II strains tested, all grew at 40 and 41°C, whereas only 3 of 15 group I strains (haplotypes B8[P], B3[K], and B15) grew at 40°C. Three strains representing haplotype B8(P) were the only group I strains that grew at 41°C. These results contribute to a better understanding of the genetic diversity of A. citrulli associated with BFB outbreaks in Brazil, and reinforce the efficiency of MLSA and PFGE analysis for assessing population structure. This study also provides the first evidence to suggest that temperature might be a driver in the ecological adaptation of A. citrulli populations.

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Comparative Analysis of Type III Secreted Effector Genes Reflects Divergence of Acidovorax citrulli Strains into Three Distinct Lineages

Phytopathology ◽

10.1094/phyto-12-13-0350-r ◽

2014 ◽

Vol 104 (11) ◽

pp. 1152-1162 ◽

Cited By ~ 24

Author(s):

Noam Eckshtain-Levi ◽

Tamar Munitz ◽

Marija Živanović ◽

Sy M. Traore ◽

Cathrin Spröer ◽

...

Keyword(s):

Comparative Analysis ◽

Type Iii Secretion ◽

Pathogenic Bacteria ◽

Citrullus Lanatus ◽

Housekeeping Genes ◽

Type Iii ◽

Effector Genes ◽

Acidovorax Citrulli ◽

Group I ◽

Group Ii

Acidovorax citrulli causes bacterial fruit blotch of cucurbits, a serious economic threat to watermelon (Citrullus lanatus) and melon (Cucumis melo) production worldwide. Based on genetic and biochemical traits, A. citrulli strains have been divided into two distinct groups: group I strains have been mainly isolated from various non-watermelon hosts, while group II strains have been generally isolated from and are highly virulent on watermelon. The pathogen depends on a functional type III secretion system for pathogenicity. Annotation of the genome of the group II strain AAC00-1 revealed 11 genes encoding putative type III secreted (T3S) effectors. Due to the crucial role of type III secretion for A. citrulli pathogenicity, we hypothesized that group I and II strains differ in their T3S effector repertoire. Comparative analysis of the 11 effector genes from a collection of 22 A. citrulli strains confirmed this hypothesis. Moreover, this analysis led to the identification of a third A. citrulli group, which was supported by DNA:DNA hybridization, DNA fingerprinting, multilocus sequence analysis of conserved genes, and virulence assays. The effector genes assessed in this study are homologous to effectors from other plant-pathogenic bacteria, mainly belonging to Xanthomonas spp. and Ralstonia solanacearum. Analyses of the effective number of codons and gas chromatography content of effector genes relative to a representative set of housekeeping genes support the idea that these effector genes were acquired by lateral gene transfer. Further investigation is required to identify new T3S effectors of A. citrulli and to determine their contribution to virulence and host preferential association.

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Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity

10.1101/2021.05.24.445476 ◽

2021 ◽

Author(s):

IRENE JIMENEZ-GUERRERO ◽

MONICA SONAWANE ◽

NOAM ECKSHTAIN-LEVI ◽

GUSTAVO M. DA SILVA ◽

FRANCISCO PEREZ-MONTANO ◽

...

Keyword(s):

Amino Acid ◽

Pseudomonas Syringae ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Variable Region ◽

Callose Deposition ◽

Type Iii ◽

Acidovorax Citrulli ◽

Group I ◽

Group Ii

Bacterial fruit blotch (BFB) is a serious disease of melon and watermelon caused by the Gram-negative bacterium Acidovorax citrulli. The strains of the pathogen can be divided into two major genetic groups, I and II. While group I strains are strongly associated with melon, group II strains are more aggressive on watermelon. Like many pathogenic bacteria, A. citrulli secretes a variety of protein effectors to the host cell via the type III secretion system. In the present study, we characterized AopW1, an A. citrulli type III-secreted effector that shares similarity with the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. aopW1 is present in group I and II strains, encoding products of 485 amino acids. Although highly conserved in most of the sequence, AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, including 14 amino acid differences between groups. Here we show that group I AopW1 is more toxic to yeast and plant cells than group II AopW1, having a stronger actin filament disruption activity, and increased ability to reduce plant callose deposition. We demonstrate the role of some of these 14 amino acid positions in determining the phenotypic differences between the two versions of the effector. Moreover, cellular analyses revealed that in addition to the interaction with actin filaments, AopW1 is localized to the endoplasmic reticulum, chloroplasts, and early and recycling plant endosomes, with differences observed between the two AopW1 versions. Finally, we show that overexpression of the endosome-associated protein EHD1 that increases cellular recycling, attenuates the toxic effects exerted by AopW1 and increases defence responses. This study provides insights into the HopW1 family of bacterial effectors and their interactions with the plant cell and provides first evidence on the involvement of EHD1 in response to biotic stress.

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Ultrastructural Lesions Produced by Alcohol and Sucrose in the Heart and Liver of C57BL Mice

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068035 ◽

1970 ◽

Vol 28 ◽

pp. 208-209

Author(s):

K.K. SEKHRI ◽

C.S. ALEXANDER ◽

H.T. NAGASAWA

Keyword(s):

Osmium Tetroxide ◽

Male Mice ◽

Alcohol Group ◽

Group Iii ◽

Group I ◽

C57bl Mice ◽

Group Ii

C57BL male mice (Jackson Lab., Bar Harbor, Maine) weighing about 18 gms were randomly divided into three groups: group I was fed sweetened liquid alcohol diet (modified Schenkl) in which 36% of the calories were derived from alcohol; group II was maintained on a similar diet but alcohol was isocalorically substituted by sucrose; group III was fed regular mouse chow ad lib for five months. Liver and heart tissues were fixed in 2.5% cacodylate buffered glutaraldehyde, post-fixed in 2% osmium tetroxide and embedded in Epon-araldite.

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Heterogeneity and Immunochemical Properties of Anti-β2-glycoprotein I Autoantibodies

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615218 ◽

1998 ◽

Vol 80 (09) ◽

pp. 393-398 ◽

Cited By ~ 33

Author(s):

V. Regnault ◽

E. Hachulla ◽

L. Darnige ◽

B. Roussel ◽

J. C. Bensa ◽

...

Keyword(s):

Fluid Phase ◽

Anticardiolipin Antibodies ◽

Dual Reactivity ◽

Group Iii ◽

Important Group ◽

Epitope Specificity ◽

Glycoprotein I ◽

Group I ◽

Group Ii ◽

Sufficient Concentration

SummaryMost anticardiolipin antibodies (ACA) associated with antiphospholipid syndrome (APS) are directed against epitopes expressed on β2-glycoprotein I (β2GPI). Despite a good correlation between standard ACA assays and those using purified human β2GPI as the sole antigen, some sera from APS patients only react in the latter. This is indicative of heterogeneity in anti-β2GPI antibodies. To characterize their reactivity profiles, human and bovine β2GPI were immobilized on γ-irradiated plates (β2GPI-ELISA), plain polystyrene precoated with increasing cardiolipin concentrations (CL/β2GPI-ELISA), and affinity columns. Fluid-phase inhibition experiments were also carried out with both proteins. Of 56 selected sera, restricted recognition of bovine or human β2GPI occurred respectively in 10/29 IgA-positive and 9/22 IgM-positive samples, and most of the latter (8/9) were missed by the standard ACA assay, as expected from a previous study. Based on species specificity and ACA results, IgG-positive samples (53/56) were categorized into three groups: antibodies reactive to bovine β2GPI only (group I) or to bovine and human β2GPI, group II being ACA-negative, and group III being ACA-positive. The most important group, group III (n = 33) was characterized by (i) binding when β2GPI was immobilized on γ-irradiated polystyrene or cardiolipin at sufficient concentration (regardless of β2GPI density, as assessed using 125I-β2GPI); (ii) and low avidity binding to fluid-phase β2GPI (Kd in the range 10–5 M). In contrast, all six group II samples showed (i) ability to bind human and bovine β2GPI immobilized on non-irradiated plates; (ii) concentration-dependent blockade of binding by cardiolipin, suggesting epitope location in the vicinity of the phospholipid binding site on native β2GPI; (iii) and relative avidities approximately 100-fold higher than in group III. Group I patients were heterogeneous with respect to CL/β2GPI-ELISA and ACA results (6/14 scored negative), possibly reflecting antibody differences in terms of avidity and epitope specificity. Affinity fractionation of 23 sera showed the existence, in individual patients, of various combinations of antibody subsets solely reactive to human or bovine β2GPI, together with cross-species reactive subsets present in all samples with dual reactivity namely groups III and II, although the latter antibodies were poorly purified on either column. Therefore, the mode of presentation of β2GPI greatly influences its recognition by anti-β2GPI antibodies with marked inter-individual heterogeneity, in relation to ACA quantitation and, possibly, disease presentation and pathogenesis.

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Treatment of venous leg ulcers with sulodexide

Phlebologie ◽

10.1055/s-0037-1621458 ◽

2003 ◽

Vol 32 (05) ◽

pp. 115-120 ◽

Cited By ~ 15

Author(s):

A. Franek ◽

H. Koziolek ◽

M. Kucharzewski

Keyword(s):

Pharmacological Treatment ◽

Healing Process ◽

Leg Ulcers ◽

Venous Ulceration ◽

Venous Leg Ulcers ◽

Group I ◽

Group Ii

SummaryAim: The study of the influence of sulodexide in the treatment of venous leg ulcers. Patients and method: 44 patients with chronic venous ulceration were randomly divided into two groups. Group I: 21 patients (ulceration area: 12.7-18.9 cm2), Group II: 23 patients (ulceration size: 12.1-20.3 cm2). Both groups were treated by using Unna’s boot. This dressing was changed every seven days until the ulcer had healed. Additionally, the patients in group II received the systemic pharmacological treatment with sulodexide. Results: After 7 weeks of treatment ulcers of seven patients (35%) from group I had healed, and 3 weeks later the ulceration of two more patients had healed completely. After further 7 weeks the ulcers of 12 patients had healed completely. Whereas in group II after 7 weeks of treatment ulceration of 16 (70%, p <0.05) patient had healed completely and after further 3 weeks the ulcers of the remaining 7 patients had healed, too. Conclusion: The use of sulodexide in patients with chronic venous leg ulcers accelerates the healing process.

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Effects of Rest and Exercise on Cardiac Blood Volume Determinations

Nuklearmedizin ◽

10.1055/s-0038-1629844 ◽

1997 ◽

Vol 36 (08) ◽

pp. 259-264

Author(s):

N. Topuzović

Keyword(s):

Radionuclide Ventriculography ◽

Left Ventricular ◽

Peak Exercise ◽

Volume Determination ◽

Group I ◽

Lv Volumes ◽

Group Ii ◽

Lv Volume

Summary Aim: The purpose of this study was to investigate the changes in blood activity during rest, exercise and recovery, and to assess its influence on left ventricular (LV) volume determination using the count-based method requiring blood sampling. Methods: Forty-four patients underwent rest-stress radionuclide ventriculography; Tc-99m-human serum albumin was used in 13 patients (Group I), red blood cells was labeled using Tc-99m in 17 patients (Group II) in vivo, and in 14 patients (Group III) by modified in vivo/in vitro method. LV volumes were determined by a count-based method using corrected count rate in blood samples obtained during rest, peak exercise and after recovery. Results: In group I at stress, the blood activity decreased by 12.6 ± 5.4%, p <0.05, as compared to the rest level, and increased by 25.1 ± 6.4%, p <0.001, and 12.8 ± 4.5%, p <0.05, above the resting level in group II and III, respectively. This had profound effects on LV volume determinations if only one rest blood aliquot was used: during exercise, the LV volumes significantly decreased by 22.1 ± 9.6%, p <0.05, in group I, whereas in groups II and III it was significantly overestimated by 32.1 ± 10.3%, p <0.001, and 10.7 ± 6.4%, p <0.05, respectively. The changes in blood activity between stress and recovery were not significantly different for any of the groups. Conclusion: The use of only a single blood sample as volume aliquot at rest in rest-stress studies leads to erroneous estimation of cardiac volumes due to significant changes in blood radioactivity during exercise and recovery.

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A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648952 ◽

1994 ◽

Vol 72 (05) ◽

pp. 745-749 ◽

Cited By ~ 7

Author(s):

Elza Chignier ◽

Maud Parise ◽

Lilian McGregor ◽

Caroline Delabre ◽

Sylvie Faucompret ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Peripheral Blood ◽

Vascular Trauma ◽

Activated Platelets ◽

Group I ◽

Group Ii ◽

Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

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