Biological Control of Fireblight (Erwinia amylovora [Burr.] Winslow et al.) on Ornamentals

Several yellow pigmented, rod-shaped bacterial strains with antagonistic activity against E. amylovora had been isolated from blighted ornamental shrubs. Criteria of the mode of action have been investigated under in-vitro conditions and in inoculation experiments. In contrast to the noninhibitory isolates of the same host origin the active forms obviously produce a substantial principle. This is active only under acid conditions, it is heat-stabile, it can be dialyzed and is solublein water and methanol. It is not a phenolic compound. In disease control experiments using culture filtrates of the antagonistic bacterial isolates fireblight could be reduced to a limited extend following shoot tip inoculation of Cotoneaster bullatus under controlled conditions. Compared to application of the living antagonists in the control experiments disease reduction, however, was considerably less expressed.     

Weather and fireblight in England

A method for assessing potential fireblight activity in the field in southeast England is described. It is based on potential doublings (PD), derived from in vitro growth rates of Erwinia amylovora at different temperatures, combined with a rain score (R) derived from daily precipitation values. Where the incubation period (I) was known, the coefficient of correlation between I/R and PD was 085 (P < 0–001). When I was regressed on PD and R, the multiple correlation coefficient was 0–97. The low level of fireblight in England precluded direct testing of the predictive value of the relationship between PD, I and R but its application to growing season weather of 1955-74 highlighted those years when fireblight activity was high in a particular host. Whilst within the range 65–86 ^oF (18–30 ^oC) there is good agreement between accumulated potential doubling values and accumulated degree days, the rain score would probably require modification in other climates.     

Phytotoxic material from associations betweenErwinia amylovora and pear tissue culture: Possible role in necrotic symptomatology of fireblight disease

Pear cell suspension cultures (PCSC) inoculated with virulent strains of the fireblight bacterium,Erwinia amylovora, exhibited massive necrosis within 7 days, whereas avirulentE. amylovora strains and other enterobacteria generally elicited very slight or no necrotic reactions. These results were generally repeated when pear seedlings (1–2 months old) were inoculated with these same bacterial strains. Fractions derived from massively necrotized PCSC were tested for biological activity against healthy PCSC and pear seedling cuttings. Of these fractions, the dialyzable, 70%-ethanol-soluble material—remaining after precipitation by ethanol of high-molecular-weight polysaccharides (“amylovorin”)—exclusively inhibited growth of pear callus, plasmolyzed and disintegrated pear cells in PCSC, and caused some browning of pear callus. Although cuttings of young pear seedlings placed in solutions containing this dialyzable, ethanol-soluble material generally became massively necrotized within 3 days, some residual methodological problems with this bioassay procedure must be solved. These observations suggest that some relatively small molecule(s) formed in PCSC inoculated with virulentE. amylovora can exert antagonistic biological activity against pear tissue and may play a role in the symptomatology characteristic of the fireblight disease.     

Fireblight in Kent, England in relation to weather (1955–1976)

A system for the assessment of potential for fireblight Erwinia amylovora activity (PFA), based on standard temperature and rainfall records, has previously been outlined. Here some recent modifications are described and the system is discussed more fully and tested for its ability to explain outbreaks of fireblight in different hosts in Kent, south-east England for the years 1955–76. In most cases, there was a satisfactory match between PFA patterns and recorded field outbreaks and incidents and it is concluded that warnings based on the system could have lessened risk of disease in some hosts in past years. In the short term (depending on the accuracy of weather forecasts) the system can be used predictively. Its use in this way is discussed together with underlying principles of some of the criteria used and possible ways in which the precision of the system might be improved.     

The utility of inflammation and platelet biomarkers in patients with acute coronary syndromes

Introduction

Thrombotic and inflammatory mechanisms are involved in the pathophysiology of acute coronary syndrome (ACS). The aim of the study was the evaluation of inflammation (white blood cells count/WBC, C-reactive protein/CRP, interleukin-6/IL-6) and platelet (platelet count/PLT, mean platelet volume/MPV, large platelet/LPLT, beta-thromboglobulin/β-TG) biomarkers in the groups of ACS patients depending on the severity of signs and symptoms and compared to controls without coronary artery disease.

Improved recovery of Erwinia amylovora-stressed cells from pome fruit on RESC, a simple, rapid and differential medium

The bacterium Erwinia amylovora causes fire blight, a serious and widespread disease of several pome fruit and ornamental plants. The use of suitable detection tools is essential for preventing its dissemination and, according to the protocol of the European and Mediterranean Plant Protection Organization, the isolation and further identification of E. amylovora is the only conclusive test of its presence. However, bacterial growth on solid media can be hampered when the pathogen is suffering stressful conditions in pome fruit or in other habitats. Since copper is an essential micronutrient that, in E. amylovora, also increases the exopolysaccharide production in rich-nutrient media, we have designed a non-selective differential medium containing 1.5 mM CuSO₄ to improve the recovery of E. amylovora from plants under unfavorable conditions. In this new medium named Recovery Erwinia amylovora-Stressed Cells (RESC), its colonies were easily distinguished by a light yellow color and a high mucus production. The plating recovery of several E. amylovora strains in vitro and from naturally infected samples was significantly improved with respect to other media routinely employed, particularly when the pathogen was suffering stressful conditions. Thus, the recovery of stressed E. amylovora cells (after UV irradiation, nutrient deprivation, or the presence of copper ions in non-copper-complexing media) was significantly enhanced on RESC medium, and their culturability period extended. Therefore, RESC is a useful and valuable medium for the isolation of E. amylovora when adverse conditions in the natural environment are expected.     

Fireblight Erwinia amylovora and weather: a comparison of warning systems

Warning systems for fireblight Erwinia amylovora developed in New York, Illinois and California, USA, and in south-east England are compared. General principles which might be applicable in the different climates were sought. The consequences of applying threshold temperature values chosen for one area in a different climatic area were examined using Sacramento, California; Rochester, New York; Vlissingen, The Netherlands; Kent, England as examples. A graded system for assessing fireblight risks, derived from all the systems, is suggested. It takes into account both risks of infection and risks of high insect activity and it is best used in conjunction with Billing's incubation period assessment system.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Proferrioxamine synthesis in Erwinia amylovora in response to precursor or hydroxylysine feeding: metabolic profiling with liquid chromatography-electrospray mass spectrometry

Metabolic profiling by capillary liquid chromatography-electrospray mass spectrometry was used to monitor shifts in the proferrioxamine profiles of Erwinia amylovora in response to externally supplied potential proferrioxamine precursors, selected stable-isotope-labeled precursors and atypical precursors. Based on the qualitative and quantitative shifts in the proferrioxamine profiles, lysine and arginine are unambiguous, and agmatine, ornithine, diaminobutyric acid and the corresponding C_3–5 diamines are highly likely precursors for proferrioxamine biosynthesis in E. amylovora. 5-Hydroxylysine (Hyl), a recently discovered growth inhibitor for E. amylovora, suppresses proferrioxamine production. The Hyl-induced growth inhibition can be reversed by basic amino acids. The basic amino acids also partly restore proferrioxamine synthesis.Part 12 in the series Metabolites of Erwinia, for Parts 10 and 11 see Feistner (1994d) and Feistner (1995b), respectively. Presented, in part, at ALEX '93. San Francisco. October 5–7. 1993, and at the 42nd ASMS Conference. Chicago. May 29–June 3, 1994.     

Production of tobacco aroma from lutein. Specific role of the microorganisms involved in the process

A mixed culture formed by Bacillus sp. and Geotrichum sp. produced tobacco aroma compounds from the carotenoid lutein through the formation of the intermediate -ionone. Both microorganisms can grow independently in a medium supplemented with lutein, but only Geotrichum produces -ionone. This intermediate was incorporated by the bacilli, converted to aroma and this product excreted to the culture medium. Bacillus sp. did not utilize -ionone for growth but modified it. We conclude that, in the bioconversion of lutein to products with tobacco aroma, Geotrichum sp. is involved in carotenoid oxidation to produce -ionone and Bacillus sp. is responsible for the norisoprenoid reduction to produce 7,8-dihydro--ionone and 7,8-dihydro--ionol.     

Selective inhibition of Erwinia amylovora by the herbicidally active germination-arrest factor (GAF) produced by Pseudomonas bacteria

Aims: The germination‐arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4‐formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. This study was undertaken to determine whether GAF has antimicrobial activity in addition to its inhibitory effects on grass seed germination. Methods and Results: Culture filtrate from Ps. fluorescens WH6 had little or no effect on 17 species of bacteria grown in Petri dish lawns, but the in vitro growth of Erwinia amylovora, the causal agent of the disease of orchard crops known as fire blight, was strongly inhibited by the filtrate. The anti‐Erwinia activity of WH6 culture filtrate was shown to be due to its GAF content, and a commercially available oxyvinylglycine, 4‐aminoethoxyvinylglycine (AVG), exhibited anti‐Erwinia activity similar to that of GAF. The effects of GAF on Erwinia were reversed by particular amino acids. Conclusions: The biological properties of GAF include a rather specific antimicrobial activity against Erw. amylovora. This may be a general property of oxyvinylglycines as AVG exhibited similar activity. The ability of particular amino acids to reverse GAF inhibition is consistent with a potential effect of this compound on the activity of aminotransferases. Significance and Impact of the Study: The results presented here demonstrate a novel antimicrobial activity of oxyvinylglycines and suggest that GAF and/or GAF‐producing bacteria may have potential for the control of fire blight.     

Complete Genome Sequence of the Plant Pathogen Erwinia amylovora Strain ATCC 49946

Erwinia amylovora causes the economically important disease fire blight that affects rosaceous plants, especially pear and apple. Here we report the complete genome sequence and annotation of strain ATCC 49946. The analysis of the sequence and its comparison with sequenced genomes of closely related enterobacteria revealed signs of pathoadaptation to rosaceous hosts.Erwinia amylovora, a plant-associated member of the Enterobacteriaceae, causes fire blight, a devastating disease of rosaceous plants, especially pear and apple (6). The complete genome of Ea273 (ATCC 49946), a virulent strain isolated from an infected apple tree in New York State, was sequenced. Total DNA was extracted and prepared in pMAQ1 shotgun libraries. The complete shotgun sequence was obtained by using dye terminator chemistry in ABI 3730 automated sequencers and contains 88,457 reads (11.12-fold coverage), yielding a theoretical coverage of the genome of 99.99%. The sequence was assembled, finished, and annotated as described previously (1, 5), using Artemis (4) to collate data and facilitate annotation.The genome of E. amylovora consists of a circular chromosome of 3,805,874 bp and two plasmids, AMYP1 (28,243 bp) and AMYP2 (71,487 bp). Coding regions in the chromosome account for 85.1% of the total sequence, with 3,483 identified coding sequences (CDS). Two hundred fifty-four (7%) of the CDSs do not have any matches in current NCBI databases; 114 (3.3%) correspond to conserved hypothetical proteins. Forty-nine CDSs (1.4%) are similar to genes from mobile elements such as integrases, transposases, and bacteriophages, and 110 CDSs (3.2%) were classified as pseudogenes due to interruptions or truncations of the CDSs. The remaining 2,956 annotated CDSs include among other categories genes involved in biosynthesis of the cellular envelope and modifications of surface proteins (299 CDSs [11%]) and genes involved in signal transduction and regulation (228 CDSs [8%]). Seven rRNA operons and 78 tRNA sequences were identified in the chromosome; two new clusters were identified (AMY1550-1575 and AMY2648-2676) that resemble the T3SS-encoding SSR-1 island of Sodalis glossinidius (2), and four clusters that contain genes for biosynthesis of flagella, which based on their location might be regulated independently.The smaller plasmid, AMYP1, had been reported as pEA29 (3); its sequence is nearly identical to the one reported here. The larger plasmid, AMYP2, renamed pEA72 for consistency in nomenclature, contains 87 predicted CDSs, with two predicted mobile-element-related CDSs and one pseudogene. Among the CDSs with annotated functions are a cluster of genes (AMYP2_49 to AMYP2_62) that encode a putative type IV fimbrial system (pil genes).The genome of E. amylovora is only 3.8 Mb long, whereas most free-living enterobacteria, including plant pathogens, have genomes of 4.5 Mb to 5.5 Mb. Comparison of the genome of Ea273 with the sequenced genomes of 15 closely related enterobacteria identified 21 lineage-specific regions, which might be considered genomic islands. E. amylovora has many more predicted pseudogenes, relative to other enterobacteria with similar lifestyles. Given its size and the preponderance of pseudogenes, genome reduction may have occurred via mutational inactivation and subsequent deletion with the following consequences: E. amylovora has fewer genes involved in anaerobic respiration and fermentation than are found in typical related enterobacteria; this likely result in a reduced capacity to live in anaerobic environments.The genome sequence of E. amylovora has revealed clear signs of pathoadaptation to the rosaceous plant environment. For example, T3SS-related proteins are present that are more similar to proteins of other plant pathogens than to proteins of closely related enterobacteria. These include type III effectors, homologous to those of plant-pathogenic pseudomonads, which confer virulence to E. amylovora in plants, and a sorbitol-metabolizing cluster that may confer a competitive advantage for survival in rosaceous plants. The reduced genome size and erosion or loss of genes involved in anaerobic respiration and nitrate assimilation are remarkable, relative to other plant- and animal-pathogenic members of the Enterobacteriaceae.     

The effect of nutrient medium and age ofErwinia amylovora andErwinia herbicola cultures on their reactivity in agglutination test

The influence of the type of nutrient medium (meat-peptone nutrient agar, KING B medium, YDC medium) and the age of bacterial cultures ofErwinia amylovora andE. herbicola on reactivity of their antigens with homologous antisera were examined. In the case ofE. amylovora, the best agglutination reactions were observed with isolates cultivated on the YDC nutrient medium. After 192 h of cultivation, seven outof ten isolates reacted positively. The reactivity ofE. herbicola antigen decreased in dependence on culture age more rapidly than withE. amylovora. The highest rates of positive agglutination reactions were observed withE. herbicola cultivated on the YDC and KING B nutrient media. After 144 h of cultivation on both these nutrient media eight out of ten isolates reacted positively.     

Signalling requirements for Erwinia amylovora‐induced disease resistance,callose deposition and cell growth in the non‐host Arabidopsis thaliana

Erwinia amylovora is the causal agent of the fire blight disease in some plants of the Rosaceae family. The non‐host plant Arabidopsis serves as a powerful system for the dissection of mechanisms of resistance to E. amylovora. Although not yet known to mount gene‐for‐gene resistance to E. amylovora, we found that Arabidopsis activated strong defence signalling mediated by salicylic acid (SA), with kinetics and amplitude similar to that induced by the recognition of the bacterial effector avrRpm1 by the resistance protein RPM1. Genetic analysis further revealed that SA signalling, but not signalling mediated by ethylene (ET) and jasmonic acid (JA), is required for E. amylovora resistance. Erwinia amylovora induces massive callose deposition on infected leaves, which is independent of SA, ET and JA signalling and is necessary for E. amylovora resistance in Arabidopsis. We also observed tumour‐like growths on E. amylovora‐infected Arabidopsis leaves, which contain enlarged mesophyll cells with increased DNA content and are probably a result of endoreplication. The formation of such growths is largely independent of SA signalling and some E. amylovora effectors. Together, our data reveal signalling requirements for E. amylovora‐induced disease resistance, callose deposition and cell fate change in the non‐host plant Arabidopsis. Knowledge from this study could facilitate a better understanding of the mechanisms of host defence against E. amylovora and eventually improve host resistance to the pathogen.     

Cardiac Overexpression of Constitutively Active Galpha q Causes Angiotensin II Type1 Receptor Activation,Leading to Progressive Heart Failure and Ventricular Arrhythmias in Transgenic Mice

Background

Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gα_q-TG) exhibt progressive heart failure and ventricular arrhythmias after the initiating stimulus of transfected constitutively active Gα_q becomes undetectable. However, the mechanisms are still unknown. We examined the effects of chronic administration of olmesartan on heart failure and ventricular arrhythmia in Gα_q-TG mice.

Methodology/Principal Findings

Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gα_q-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan administration prevented the severe reduction of left ventricular fractional shortening, and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gα_q-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gα_q-TG but in none of 10 olmesartan-treated Gα_q-TG. The collected QT interval and monophasic action potential duration in the left ventricle were significantly shorter in olmesartan-treated Gα_q-TG than in vehicle-treated Gα_q-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased and olmesartan significantly decreased the expression of these genes in Gα_q-TG mouse ventricles. The expression of canonical transient receptor potential (TRPC) 3 and 6 channel and angiotensin converting enzyme (ACE) proteins but not angiotensin II type 1 (AT₁) receptor was increased in Gα_q-TG ventricles compared with NTG mouse ventricles. Olmesartan significantly decreased TRPC6 and tended to decrease ACE expressions in Gα_q-TG. Moreover, it increased AT₁ receptor in Gα_q-TG.

Conclusions/Significance

These findings suggest that angiotensin II type 1 receptor activation plays an important role in the development of heart failure and ventricular arrhythmia in Gα_q-TG mouse model of heart failure.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Expression of firefly luciferase gene in Erwinia amylovora

In this study we describe an ef?cient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the ?re?y luciferase gene of Photinus pyralis, which is further controlled by IPTG‐inducible promoter. Stably transformed E. amylovora cells maintain the same infectivity as the wild‐type strain and, after induction with IPTG, produce luciferase. Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL). The transformed E. amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post‐infection. Our ?ndings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.     

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non‐host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild‐type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR‐deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease‐specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight‐infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.     

A method to produce encapsulatable units for synthetic seeds in Asparagus officinalis

A method to produce encapsulatable units for synthetic seeds was developed in Asparagus officinalis L. Encapsulatable units with high conversion ability in non-sterile soil were produced from somatic embryos by a pre-encapsulation culture. The synthetic seeds containing somatic embryos without the pre-encapsulation culture did not germinate in soil. When the pre-encapsulation culture medium did not contain growth regulators, the roots elongated too much to accomplish encapsulation. Several growth regulators were studied and indole-3-acetic acid was considered to be optimum at 28.5 M. The pre-encapsulation culture medium with indole-3-acetic acid inhibited the growth of roots during the pre-encapsulation culture and produced compact encapsulatable units. The growth of roots was promoted when plants were produced from the encapsulatable units. The percent conversion of the synthetic seeds with these encapsulatable units was 72% in non-sterile soil. This is the first report on synthetic seeds in Asparagus officinalis L.