Amylovora Infection Research Articles

Biological control of fire blight of apple and pear with antagonistic Lactobacillus plantarum

Lactic acid bacteria (LAB) can be a source of biological control agents (BCA) of fire blight disease. Several species of LAB are inhabitants of plants and are currently used as biopreservatives of food because of their antagonistic properties against bacteria, and are considered as generally safe. Candidates to BCA were selected from a large collection of LAB strains obtained from plant environments. Strains were first chosen based on the consistency of the suppressive effect against E. amylovora infections in detached plant organs (flowers, fruits and leaves). Lactobacillus plantarum strains PC40, PM411, TC54 and TC92 were effective against E. amylovora in most of the experiments performed. Besides, strains PM411, TC54 and TC92 had strong antagonistic activity against E. amylovora and also other target bacteria, and presented genes involved in plantaricin biosynthesis (plnJ, plnK, plnL, plnR and plnEF). The strains efficiently colonized pear and apple flowers; they maintained stable populations for at least 1 week under high RH conditions, and survived at low RH conditions. They were effective in preventing fire blight on pear flowers, fruits and leaves, as well as in whole plants and in a semi-field blossom assay. The present study confirms the potential of certain strains of L. plantarum to be used as active ingredient of microbial biopesticides for fire blight control that could be eventually extended to other plant bacterial diseases.

EDS1 Contributes to Nonhost Resistance of Arabidopsis thaliana Against Erwinia amylovora

Erwinia amylovora causes fire blight in rosaceous plants. In nonhost Arabidopsis thaliana, E. amylovora triggers necrotic symptoms associated with transient bacterial multiplication, suggesting either that A. thaliana lacks a susceptibility factor or that it actively restricts E. amylovora growth. Inhibiting plant protein synthesis at the time of infection led to an increase in necrosis and bacterial multiplication and reduced callose deposition, indicating that A. thaliana requires active protein synthesis to restrict E. amylovora growth. Analysis of the callose synthase-deficient pmr4-1 mutant indicated that lack of callose deposition alone did not lead to increased sensitivity to E. amylovora. Transcriptome analysis revealed that approximately 20% of the genes induced following E. amylovora infection are related to defense and signaling. Analysis of mutants affected in NDR1 and EDS1, two main components of the defense-gene activation observed, revealed that E. amylovora multiplied ten times more in the eds1-2 mutant than in the wild type but not in the ndr1-1 mutant. Analysis of mutants affected in three WRKY transcription factors showing EDS1-dependent activation identified WRKY46 and WRKY54 as positive regulators and WRKY70 as a negative regulator of defense against E. amylovora. Altogether, we show that EDS1 is a positive regulator of nonhost resistance against E. amylovora in A. thaliana and hypothesize that it controls the production of several effective defenses against E. amylovora through the action of WRKY46 and WRKY54, while WRKY70 acts as a negative regulator.

Pome fruit health

The pome fruits, apple and pear, represent very important fruit crops in Europe. They are grown on 490,000 ha in 15 countries with a production of more than 11 million tons per year (FAO, 2003). This corresponds to an estimated annual value of €20 billion at consumer prices. European consumers are highly concerned about health and safety aspects of food. High quality fruits in sufficient amounts can only be produced on healthy plants. Thus, one of the most important tasks in crop production is an effective and sustainable management of plant diseases. Two of the most important diseases in European pome fruit growing are fire blight which is caused by the bacterium Erwinia amylovora, and the scab on apple and pear caused by the fungi Venturia inaequalis and V. pirina, respectively. Erwinia species cause not only severe losses in apple and pear but also attack numerous native wild species and popular ornamentals of the Rosaceae. Intensive investigations on these pome fruit diseases revealed new insights into the molecular aspects of the host–pathogen interactions, into possible mechanisms of defence by the host plants, into the genetic background of plant resistance and tolerance and into the molecular basis of virulence. Nowadays, nearly all European countries are facing similar problems regarding plant protection in pome fruit growing. Thus, co-operation at the international level is required and a network of more than 200 European scientists from 25 countries was established by the COST action 864 ‘Combining traditional and advanced strategies for plant protection in pome fruit growing’ (Acronym POME FRUIT HEALTH). The action focused on different aspects includes the (i) investigation of plant–pathogen interactions, (ii) germplasm resources and breeding, (iii) production methods and (iv) biotechnological resources. This special issue of Trees provides an overview of the fruitful yield of the interactions among European experts. A series of reviews and original articles cover actual research topics mainly in the fields of germplasm resources and breeding, host–pathogen interactions and fruit tree protection. For breeding, it is crucial to identify germplasm with heritable disease resistance. The development of efficient markers is essential for marker-assisted selection which is already used in breeding programs for scab resistance but is also attempted for fire blight resistance. Resource evaluation must include the susceptibility to fire blight (E. amylovora) and scab (V. inaequalis, V. pirina), but also to other diseases such as powdery mildew (Podosphaera leucotricha), pear rust (Gymnosporangium sabinae), brown spot of pear (Stemphylium vesicarium) and storage diseases (e.g. Penicillium expansum and Monilinia spec.). The knowledge of the genes behind a resistance and of its mode of action improves breeding success and offers prospects in gene technology. Recent advances are reviewed for the Vf resistance of apples against the scab. The possible involvement of secondary plant metabolites such as phenolic compounds in the defence of V. inaequalis is still debated. Various floral traits that may play a role in the onset and progression of E. amylovora infection are compared between susceptible and tolerant apple and pear varieties and the expression pattern of stress-related genes in E. amylovora-infected pear leaves is shown. It is not the core scope of Trees to deal with bacteria. However, it completes the examination of fire blight management. For progress in pome fruit protection against this bacterial D. Treutter (&) Unit Fruit Science, Technische Universitat Munchen, Durnast 2, 85354 Freising, Germany e-mail: dieter.treutter@wzw.tum.de

Rootstock-regulated gene expression patterns associated with fire blight resistance in apple.

BackgroundDesirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response.ResultsRootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population.ConclusionsRootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K 1KLFKKILKF 10L-NH 2 (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH 2 (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.

ERWINIA AMYLOVORA INFECTION OF FLOWERS AND SHOOTS IN APPLE TREES TREATED WITH PROHEXADIONE-CA

ERWINIA AMYLOVORA INFECTION OF FLOWERS AND SHOOTS IN APPLE TREES TREATED WITH PROHEXADIONE-CA

Development of a strain-specific quantitative method for monitoring Pseudomonas fluorescens EPS62e, a novel biocontrol agent of fire blight

Pseudomonas fluorescens EPS62e has been selected in a screening procedure for its high efficacy controlling Erwinia amylovora infections in flowers, immature fruits and young pear plants. We developed two monitoring methods which allowed specific detection and quantification of EPS62e by combining classical microbiological techniques with molecular tools. RAPD and unspecific-PCR fingerprints were used to differentiate EPS62e from other P. fluorescens strains. Differential amplified fragments from EPS62e were sequence characterized as SCAR markers and two primer pairs were designed and selected for their specificity against EPS62e. A SCAR primer pair was evaluated and validated for the assessment of population dynamics of EPS62e on pear plants under greenhouse conditions using plating and most probable number assays coupled to PCR. Both techniques were useful in monitoring the biological control agent. The population level of EPS62e after treatment was 7 log CFU (g f.w.) −1, which in turn decreased progressively to 4–5 log CFU (g f.w.) −1 after 17 days and then remained stable until the end of the assay 11 days later. The limit of detection of both monitoring methods developed was around 3 log CFU (g f.w.) −1, thus, providing a reliable tool for the analysis of EPS62e in greenhouse or field trials, and the assessment of threshold population levels for efficient biocontrol of fire blight.

Effect of Erwinia amylovora infection on peroxidase enzyme activity in resistant apple cultivars

Two apple cultivars that display enhanced resistance to fire blight (causal agent: Erwinia amylovora) were selected. The aim of the present study was to characterize the peroxidase (POD) enzyme activity of `Szemes alma' (a historical cultivar) and MR-03, (a Hungarian multiresistant hybrid of 'Prima') and compare them to susceptible 'Jonathan M 40' and resistant 'Remo' controls. Peroxides enzyme activity during E. amylovora infections was investigated in artificially infected apple shoots. Increases in enzyme activities were observed in a `Jonathan M40' and in 'Remo', MR-03, `Szemes alma' cultivars. There was a consistent relationship between total enzyme activity and fire blight disease severity. High activity of the peroxidase was positively correlated with the degree of resistance to fire blight. A general hypothesis that POD activity is related to fire blight susceptibility/resistance is supported by our results.

Effect of Erwinia amylovora infection on peroxidase enzyme activity in resistant apple cultivars

Two apple cultivars that display enhanced resistance to fire blight (causal agent: Erwinia amylovora) were selected. The aim of the present study was to characterize the peroxidase (POD) enzyme activity of `Szemes alma' (a historical cultivar) and MR-03, (a Hungarian multiresistant hybrid of 'Prima') and compare them to susceptible 'Jonathan M 40' and resistant 'Remo' controls. Peroxides enzyme activity during E. amylovora infections was investigated in artificially infected apple shoots. Increases in enzyme activities were observed in a `Jonathan M40' and in 'Remo', MR-03, `Szemes alma' cultivars. There was a consistent relationship between total enzyme activity and fire blight disease severity. High activity of the peroxidase was positively correlated with the degree of resistance to fire blight. A general hypothesis that POD activity is related to fire blight susceptibility/resistance is supported by our results.

Survival of Apple Rootstocks to Natural Infections of Fire Blight

Survival of replicated rootstock plantings of apple trees (Malus ×domestica) to fire blight (Erwinia amylovora) infection shows that a wide range of rootstock susceptibility exists. Trees on `Malling 26' (M.26), `Malling 9' (M.9), and `Mark' consistently had significant losses. Of the dwarfing rootstocks widely available commercially, `Budagovsky 9' (B.9) survived well with productive trees, but was not resistant to fire blight infection. The following experimental rootstocks had good survivability with many live productive trees in one or more trials: `Poland 2' (P.2), `Vineland 1' (V.1), `Malling 27 EMLA' (M.27 EMLA), `Budagovsky 491' (B.491), `Budagovsky 409' (B.409), `Vineland 7' (V.7), `Vineland 4' (V.4), and `Oregon Rootstock 1' (OAR1).

Erwinia amylovora: the molecular basis of fireblight disease

Summary Taxonomy: Bacteria; Proteobacteria; gamma subdivision; order Enterobacteriales; family Enterobacteriaceae; genus Erwinia. Microbiological properties: Gram-negative, motile rods. Related species:E. carotovora (soft-rot diseases), E. chrysanthemi (soft-rot diseases), E. (Pantoea) stewartii (Stewart's wilt of corn), E. (Pantoea) herbicola (epiphyte). Affects rosaceous plants, primarily members of the Pomoideae. Economically important hosts are apple and pear. The commercial implications of fireblight outbreaks are aggravated by the limited effectiveness of current control measures. Disease symptoms:E. amylovora infection is characterized by water soaking of infected tissue, followed by wilting and tissue necrosis. Necrosis gives tissue a scorched, blackened appearance, giving rise to the name fireblight. Symptoms are often localized to blossom bracts or young shoots but, in highly susceptible hosts, can spread systemically resulting in death of the entire tree. Infections can vary in severity depending on climatic conditions and host susceptibility. Useful web site:http://www.agric.gov.ab.ca.

A Severe Outbreak of Fire Blight in Woody Ornamental Rosaceae Plants in North Florida and South Georgia.

Woody ornamentals including 'Bradford' and 'Aristocrat' pears (Pyrus calleryana), Indian hawthorn (Rhaphiolepis sp.), and cotoneaster (Cotoneaster spp.) developed blossom and shoot blight symptoms suggestive of fire blight caused by Erwinia amylovora. Disease was observed in several counties in north Florida and south Georgia and samples were sent to Florida Extension Plant Diagnostic Clinic-Quincy (FEPDC-Q). The incidences in individual 'Bradford' pear were as high as 30 to 40 strikes per tree. This level of severe E. amylovora infections on 'Bradford' pear has not been seen in Florida for the last 10 years based on FEPDC-Q data. Symptomatic plants first appeared in the landscape and nurseries around the first week of April 2000 in the following counties: Bay, Holmes, Washington, Gadsden, Leon, Jefferson (north Florida), and Decatur (south Georgia). Severely infected 'Bradford' pear trees were also observed in Georgia cities of Cairo, Thomasville, Moultrie, and Tifton. The presence of E. amylovora in samples was verified by pEA29-polymerase chain reaction (PCR) (2,4), fatty acid methyl ester analysis (MIDI, Newark, DE), and colony type on semi-selective modified Miller-Schroth (MMS) medium (3). Of the 16 samples tested by PCR, E. amylovora was present in all. Actively growing shoots of Cotoneaster salicifolius (a susceptible host) were inoculated with a bacterium isolated from 'Bradford' pears that was identified as E. amylovora by PCR. Dark brown necrotic tissues with typical fire blight appearance developed within 10 days. E. amylovora was reisolated on MMS medium from inoculated shoot tissues of C. salicifolius and confirmed by PCR. The presence of E. amylovora from ornamental pears, Indian hawthorn, and cotoneasters was previously reported in Florida (1). Cool spring temperatures in the Florida panhandle and several rain events during the bloom period may have stimulated the severe outbreak of fire blight. This severe outbreak highlights the importance of using a forecasting model such as Maryblyt 4.3 to predict unexpected infection periods so that preventive control measures can be taken.

Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple

A cDNA clone of the gene encoding attacin was used to construct three plasmid binary vectors in which attE was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream B domain (35S) (p35SAtt), 35S with the untranslated leader sequence of alfalfa mosaic virus RNA 4 (AMV) (p35SAMVAtt), and 35S with AMV and the signal peptide of pathogenesis-related protein 1b from tobacco (SP) (p35SAMVSPAtt), respectively. These plasmids and pLDB15 containing attE under the control of the potato proteinase inhibitor II (Pin2) promoter were used in Agrobacterium-mediated transformation of the apple scion cultivar `Galaxy' and the apple rootstock M.26 to enhance resistance to Erwinia amylovora, the bacterium that causes fire blight. The mean attacin content of transgenic lines containing attacin with AMV was three times higher than lines without AMV. Northern blots suggested that AMV functioned in apple as it does in other plant species by enhancing translation of attE mRNA. Transgenic `Galaxy' lines with attacin fused to SP had lower attacin content than lines without SP. In vitro assays indicated that attacin was partially degraded in the intercellular fluid of apple leaves. However, transgenic `Galaxy' lines transformed with attacin fused to SP had significantly less disease than those without SP suggesting that intercellularly secreted attacin is more effective in reducing E. amylovora infection than intracellularly localized attacin. A negative correlation was observed between attacin content and disease resistance in Pin2Att transgenic `Galaxy' lines following inoculation with E. amylovora, suggesting that attacin enhances resistance to fire blight.

Comparison ofmalusinfection by the pathogenerwinia amylovoraand colonisation by the saprophyteerwinia herbicolaby electron microscopy

Abstract Erwinia amylovora infection of either apple stem or leaf tissue resulted in xylem occlusion by a fibrillar matrix material, determined by Thiery’ s thiocarbohydrazide (TCH) test to be the extracellular polysaccharide from E. amylovora. Plasmolysis of the xylem‐parenchyma was also detected and was most likely a result of a toxin produced by the pathogen. This plasmolysis resulted in the collapse and death of the cell. An Erwinia herbicola isolate which had previously expressed inhibitory effects on E. amylovora was contained at the site of inoculation.