Role of antibiotic production by Erwinia herbicola Eh252 in biological control of Erwinia amylovora.

Erwinia herbicola Eh252 is a nonpathogenic epiphytic bacterium that reduces fire blight incidence when sprayed onto apple blossoms before inoculation with Erwinia amylovora, the causal agent of fire blight. Eh252 was found to produce on minimal medium an antibiotic that inhibited the growth of E. amylovora. This antibiotic was inactivated by histidine but not by Fe(II), was sensitive to proteolytic enzymes, and showed a narrow host range of activity. To determine the role of this antibiotic in the control of fire blight, two prototrophic Tn5-induced mutants, 10:12 and 17:12, that had lost their ability to inhibit E. amylovora on plates (Ant- mutants) were compared with the wild-type strain for their ability to suppress fire blight in immature pear fruits. The two mutants had single Tn5 insertions in the chromosome; although they grew in immature pear fruits at a rate similar to that of the wild-type strain, neither of these mutants suppressed fire blight as well as Eh252 did. The Tn5-containing fragment isolated from 10:12 was used to mutagenize Eh252 by marker exchange. Derivatives that acquired the Tn5-containing fragment by homologous recombination lost the ability to inhibit E. amylovora on minimal medium. Furthermore, the three Ant- derivatives tested were also affected in their ability to inhibit E. amylovora in immature pear fruits. The results obtained suggest that antibiotic production is a determinant of the biological control of E. amylovora by Eh252, but that another mechanism(s) is involved.     

Application of the Maryblyt Model for the Infection of Fire Blight on Apple Trees at Chungju,Jecheon, and Eumsung during 2015–2020

To preventively control fire blight in apple trees and determine policies regarding field monitoring, the Maryblyt ver. 7.1 model (MARYBLYT) was evaluated in the cities of Chungju, Jecheon, and Eumseong in Korea from 2015 to 2020. The number of blossom infection alerts was the highest in 2020 and the lowest in 2017 and 2018. And the common feature of MARYBLYT blossom infection risks during the flowering period was that the time of BIR-High or BIR-Infection alerts was the same regardless of location. The flowering periods of the trees required to operate the model varied according to the year and geographic location. The model predicts the risk of “Infection” during the flowering periods, and recommends the appropriate times to control blossom infection. In 2020, when flower blight was severe, the difference between the expected date of blossom blight symptoms presented by MARYBLYT and the date of actual symptom detection was only 1–3 days, implying that MARYBLYT is highly accurate. As the model was originally developed based on data obtained from the eastern region of the United States, which has a climate similar to that of Korea, this model can be used in Korea. To improve field utilization, however, the entire flowering period of multiple apple varieties needs to be considered when the model is applied. MARYBLYT is believed to be a useful tool for determining when to control and monitor apple cultivation areas that suffer from serious fire blight problems.     

Characterization of Bacillus strains from apple and pear trees in South Africa antagonistic to Erwinia amylovora

In order to find reasons for the absence of fire blight in most countries of the Southern hemisphere, bark samples from apple and pear trees in orchards of the Western Cape region in South Africa were extracted for bacteria which could be antagonistic to Erwinia amylovora. Screening was done in the late growth season and mainly Gram-positive bacteria were isolated. Approximately half of them produced growth inhibition zones on a lawn of E. amylovora. Most isolates were classified as Bacillus megaterium by microbiological assays and in API 50 test systems. They were visualized in the light microscope as non-motile large rods. These strains may not be responsible for the absence of fire blight in orchards, but they may indicate unfavourable climatic conditions for Gram-negative bacteria including E. amylovora. They may reduce the ability of E. amylovora to establish fire blight and could also be useful for application in biological disease control.     

Reduction in bacterial ooze formation on immature fruitlets after preventive treatments of Fosethyl-Al against fire blight Erwinia amylovora

Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is a very important bacterial disease on apple and pear orchards with devastating effects in some production area and in some years. Fire blight control consists in a whole strategy of measures that should start with control measures in and around the fruit tree nurseries. Only the use of Vacciplant (Laminarin), an inducer of the self-defence mechanism, is registered in Belgium since 2009. In other European countries Fosethyl-Al has been registered for fire blight control. Recently, research trials have been done at Pcfruit research station for several years on the activity of ALiette (fosethyl-Al) against fire blight. Fosethyl-Al, also a plant defence enhancing molecule, applied preventively 3 times at a dose of 3.75 kg/ha standard orchard (3 x 3000 g a.i./ha standard orchard), showed a reduction in the host susceptibility and decreased the disease development on artificial inoculated flower clusters and shoots. Also a clear reduction in the ooze droplet formation on artificially inoculated immature fruitlets has been observed with this molecule. This reduction in the bacterial ooze formation is considered as a very important factor in the spread of the disease in the orchard.     

The influence of antibiotic production and pre-emptive colonization on the population dynamics of Pantoea agglomerans (Erwinia herbicola) Eh1087 and Erwinia amylovora in planta

Stigma colonization by Erwinia amylovora is the crucial first step in the development of most fire blight infections in apple and pear trees. Suppression at this point of the disease process by antagonists of E. amylovora, such as Pantoea agglomerans (Erwinia herbicola) strain Eh1087, is a rational approach to control fire blight. We tested the hypothesis that the ability of E. amylovora to compete with Eh1087 for colonization of a stigma is reduced by the potential for Eh1087 to produce the phenazine antibiotic, d-alanylgriseoluteic acid (AGA). In competition experiments on the stigmas of apple flowers, E. amylovora was significantly less successful against Eh1087 (AGA+) than against EhDeltaAGA (AGA-). Further experiments to test the importance of pre-emptive colonization of the stigma by either the pathogen or the antagonist suggested that AGA production significantly enhanced the competitiveness of Eh1087 when it was applied at the same time or 24 h before the pathogen. We also found that pre-emptive stigma colonization by either the pathogen or the antagonist resulted in a population that was resilient to subsequent invasion by a second species suggesting that niche exclusion has a dominant influence on the dynamics of bacterial populations on stigmas.     

First report on the presence of fire blight resistance in linkage group 11 ofPyrus ussuriensis Maxim

Fire blight, caused by the gram-negative bacteriumErwinia amylovora (Burrill) Winslow et al., is a dangerous disease of pome fruits, including pear. A pear breeding program for fire blight resistance was initiated in 2003 at the Department of Pomology, Warsaw University of Life Sciences, Poland. Since several Asian species are considered to be potential sources of resistance to fire blight, the susceptiblePyrus communis ‘Doyenne du Comice’ was crossed with the resistantP. ussuriensis. The F₁ full-sib progeny composed of 155 seedlings was tested for susceptibility to fire blight by artificial shoot inoculation. A framework linkage map of both parents was constructed based on 48 AFLP and 32 SSR markers and covered a length of 595 cM and 680 cM in ‘Doyenne du Comice’ andP. ussuriensis, respectively. For the first time a putative QTL for fire blight resistance inP. ussuriensis linkage group 11 was identified. Another putative QTL in linkage group 4 of ‘Doyenne du Comice’ seems to indicate that sources of fire blight resistance can be identified also in the susceptible cultivars.     

Biological Control of Fire Blight of Hawthorn (Crataegus monogyna) with Fluorescent Pseudomonas spp. under Protected Conditions

Naturally-occurring epiphytic fluorescent pseudomonads were isolated and characterized in terms of their potential to control fire blight infection of hawthorn, caused by Erwinia amylovora. Preliminary testing and selection of antagonists using an immature pear fruit assay gave some inconsistency in the amount of pathogen suppression on the pear tissue and also in the prediction of biocontrol effectiveness on the intact plant. Selected antagonists provided significant but variable control of fire blight under protected (polythene tunnel and glasshouse) conditions, with isolates HL83 and HL99 giving control of both blossom-blight and shoot-blight. In some cases the degree of control was equal to that of chemical treatments, including agrimycin 17 and experimental bactericides, and was achieved without any numerical advantage of applied control agent over pathogen. The timing of pseudomonad application in relation to pathogen inoculation was found to have a significant effect on the level of control of blossom-blight.     

Complete Genomic Sequence of Erwinia amylovora Phage PhiEaH2

Erwinia amylovora is the causative agent of fire blight, a serious disease of some Rosaceae plants. The newly isolated bacteriophage PhiEaH2 is able to lyse E. amylovora in the laboratory and has reduced the occurrence of fire blight cases in field experiments. This study presents the sequenced complete genome and analysis of phage PhiEaH2.     

Screening for fire blight resistance in apple (Malus pumila) using excised leaf assays from in vitro and in vivo grown material

The apple cultivars Greensleeves and Novole and somaclones derived from Greensleeves were tested for resistance to fire blight (Erwinia amylovora) using excised leaf bioassays. Cv. Greensleeves was consistently susceptible to fire blight in in vitro, and in glasshouse and excised leaf bioassays whilst cv. Novole was resistant. Greensleeves somaclones, identified as having intermediate levels of fire blight resistance in glasshouse and in vitro screening tests, also showed intermediate levels of resistance in excised leaf bioassays. The length of leaves and the inoculum concentration employed affected the severity of symptoms observed. Excised leaf bioassays were unsuitable as tests for fire blight resistance using field grown material.     

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 10⁴ cfu/ml and 10⁷ cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.     

Growth inhibition of Erwinia amylovora and related Erwinia species by neutralized short-chain fatty acids

Short-chain fatty acids (SCFAs) are used to preserve food and could be a tool for control of fire blight caused by Erwinia amylovora on apple, pear and related rosaceous plants. Neutralized acids were added to buffered growth media at 0.5–75 mM and tested at pHs ranging from 6.8 to 5.5. Particularly at low pH, SCFAs with a chain length exceeding that of acetic acid such as propionic acid were effective growth inhibitors of E. amylovora possibly due to uptake of free acid and its intracellular accumulation. We also observed high inhibition with monochloroacetic acid. An E. billingiae strain was as sensitive to the acids as E. amylovora or E. tasmaniensis. Fire blight symptoms on pear slices were reduced when the slices were pretreated with neutralized propionic acid. Propionic acid is well water soluble and could be applied in orchards as a control agent for fire blight.     

QTL mapping of fire blight resistance in apple

Fire blight caused by the bacterium Erwinia amylovora is a severe threat to apple and pear orchards worldwide. Apple varieties exhibit a wide range of relative susceptibility/tolerance to fire blight. Although, no monogenic resistance against fire blight has been identified yet, recent evidence indicates the existence of quantitative resistance. Potential sources of fire blight resistance include several wild Malus species and some apple cultivars. F1 progenies of ‘Fiesta’בDiscovery’ were inoculated with the Swiss strain Ea 610 and studied under controlled conditions to identify quantitative trait loci (QTLs) for fire blight resistance. Disease was evaluated at four time points after inoculation. Shoot lesion length and the area under disease progress curve (AUDPC) values were used for QTL analysis. One significant (LOD score of 7.5–8.1, p<0.001) QTL was identified on the linkage group 7 of ‘Fiesta’ (F7). The F7 QTL explained about 37.5–38.6% of the phenotypic variation.     

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.     

A QTL detected in an interspecific pear population confers stable fire blight resistance across different environments and genetic backgrounds

Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., is one of the most serious diseases of pear. The development of pear cultivars with a durable resistance is extremely important for effective control of fire blight and is a key objective of most pear breeding programs throughout the world. We phenotyped seedlings from the interspecific pear population PEAR3 (PremP003, P. × bretschneideri × P. communis) × ‘Moonglow’ (P. communis) for fire blight resistance at two different geographic locations, in France and New Zealand, respectively, employing two local E. amylovora isolates. Using a genetic map constructed with single nucleotide polymorphism (SNP) and microsatellite (SSR) markers previously developed for this segregating population, we detected a major quantitative trait locus (QTL) on linkage group (LG)2 of ‘Moonglow’ (R ² = 12.9–34.4 %), which was stable in both environments. We demonstrated that this QTL co-localizes with another major QTL for fire blight resistance previously detected in ‘Harrow Sweet’ and that the two favorable (i.e., resistant) alleles were not identical by descent. We also identified some smaller effect (R ² = 8.1–14.8 %) QTLs derived from the susceptible parent PEAR3. We propose SNP and SSR markers linked to the large effect QTL on LG2 as candidates for marker-assisted breeding for fire blight resistance in pear.     

Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.     

The role of epiphytic Pseudomonas and Pantoea population diversity on prevalence of fire blight disease

Several microbial populations on plants interact with each other and their host through the actions of secreted metabolites. However, the role of diverse microorganisms and their metabolites on plant health has yet to be fully appreciated. Here, we investigated the population diversity of two dominant epiphytic bacterial genuses in different area and their role in biological control of fire blight disease. To do so, we isolated and calculated population diversity of different Pantoea spp. and Pseudomonas spp. using serial dilution methods. The growth inhibition of Erwinia amylovora in vitro by some of these bacteria indicated the ecological significance of secondary metabolites produced by these bacteria and discuss how they might contribute to biological control of fire blight disease. Although, we did not work on the ability of these bacteria on induction of disease resistance but it could be considered for future, because they represent very different but important types of secondary metabolites. We also described how the weather conditions in different geographical regions can effect on the population of these epiphytic bacterial phenotypes leading to plant health promotion. In conclusion, we demonstrated the role of Pantoea and Pseudomonas population diversity on prevalence of fire blight in different area of north-east of Iran.     

Expression of a bacterial effector,harpin N,causes increased resistance to fire blight in <Emphasis Type="Italic">Pyrus communis</Emphasis>

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. In order to create novel mechanisms for fire blight resistance in pear, we have generated transgenic pears expressing the elicitor harpin N_ea from Erwinia amylovora under the control of the constitutive promoter CaMV35S. The transient expression of hrpN _Ea in pear cells did not provoke any apparent damage. Therefore, stable constitutive expression of hrpN _Ea was studied in seventeen transgenic clones of the very susceptible cultivar “Passe Crassane.” Most transgenic clones displayed significant reduction of susceptibility to fire blight in vitro when inoculated by E. amylovora, which was positively correlated to their degree of expression of the transgene hrpN _Ea . These results indicate that ectopic expression of a bacterial elicitor such as harpin N_ea is a promising way to improve pear resistance to fire blight.