Control Of Fire Blight Research Articles

Control of fire blight (Erwinia amylovora) on apple trees with trunk-injected plant resistance inducers and antibiotics and assessment of induction of pathogenesis-related protein genes.

Management of fire blight is complicated by limitations on use of antibiotics in agriculture, antibiotic resistance development, and limited efficacy of alternative control agents. Even though successful in control, preventive antibiotic sprays also affect non-target bacteria, aiding the selection for resistance which could ultimately be transferred to the pathogen Erwinia amylovora. Trunk injection is a target-precise pesticide delivery method that utilizes tree xylem to distribute injected compounds. Trunk injection could decrease antibiotic usage in the open environment and increase the effectiveness of compounds in fire blight control. In field experiments, after 1–2 apple tree injections of either streptomycin, potassium phosphites (PH), or acibenzolar-S-methyl (ASM), significant reduction of blossom and shoot blight symptoms was observed compared to water injected control trees. Overall disease suppression with streptomycin was lower than typically observed following spray applications to flowers. Trunk injection of oxytetracycline resulted in excellent control of shoot blight severity, suggesting that injection is a superior delivery method for this antibiotic. Injection of both ASM and PH resulted in the significant induction of PR-1, PR-2, and PR-8 protein genes in apple leaves indicating induction of systemic acquired resistance (SAR) under field conditions. The time separating SAR induction and fire blight symptom suppression indicated that various defensive compounds within the SAR response were synthesized and accumulated in the canopy. ASM and PH suppressed fire blight even after cessation of induced gene expression. With the development of injectable formulations and optimization of doses and injection schedules, the injection of protective compounds could serve as an effective option for fire blight control.

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

Preparatory work for the pest categorisation of three bacteria listed in the annexes of the Council directive 2000/29/EC Erwinia amylovora Winslow et al. Xylophilus ampelinus (Panagopoulos) Willems et al. Clavibacter michiganensis subsp. insidiosus(McCulloch) Davis et al

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.

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.

Evaluation of Strategies for Fire Blight Control in Organic Pome Fruit Without Antibiotics.

Apple and pear produced organically under the U.S. National Organic Program (NOP) standard can be treated with antibiotics for suppression of fire blight caused by Erwinia amylovora. Recent regulatory actions by the NOP, however, have lessened the likelihood of antibiotic use after the 2014 season. In response, western U.S. organic apple and pear stakeholders identified two immediate-need research objectives related to fire blight control: development of effective non-antibiotic control programs based on combinations of registered biological products; and, in apple, integration of these products with lime sulfur, which is sprayed at early bloom to reduce fruit load. In orchard trials in Oregon, increasing the frequency of treatment with biological products improved suppression of floral infection. In apple, fruit load thinning with 2% lime sulfur plus 2% fish oil (LS+FO) at 30 and 70% bloom significantly (P ≤ 0.05) reduced the proportion of blighted flower clusters in four of five orchard trials. Moreover, lime sulfur significantly (P ≤ 0.05) suppressed epiphytic populations of E. amylovora after their establishment on apple flowers. Over four trials, treatment with Aureobasidium pullulans (Blossom Protect) after LS+FO reduced the incidence of fire blight by an average of 92% compared with water only; this level of control was similar to treatment with streptomycin. In three seasons, a spray of a Pantoea agglomerans product after the 70% bloom treatment of LS+FO established the antagonist on a significantly (P ≤ 0.05) higher proportion of flowers compared with a spray of this bacterium before the thinning treatment. Consequently, in apple, biological treatments for fire blight control are not advised until after lime sulfur treatments for fruit load thinning are completed.

Potent and Specific Bactericidal Effect of Juglone (5-Hydroxy-1,4-naphthoquinone) on the Fire Blight Pathogen Erwinia amylovora

A screening of plant quinones for inhibiting effects on the bacterial fire blight pathogen Erwinia amylovora was performed. The most active compound, juglone from walnuts, has a potent and specific bactericidal effect on E. amylovora and minimal inhibitory concentrations of only 2.5-10 μM, with stronger effects at lower, but still physiological, pH values. In vitro tests with juglone and inoculated flowers of apple (Malus domestica) showed an efficacy of 67% in preventing infection. In two years of field tests juglone had variable degrees of efficacy ranging from 40 to 82%, seemingly due to environmental conditions. A phytotoxic reaction to juglone, which is known for its allelopathic effect on plants, was restricted to browning of petals; later fruit russeting was not observed. Juglone is a promising candidate for the development of a new environmentally friendly plant protectant to replace the antibiotic streptomycin currently used in fire blight control.

The undiscovered potential of dehydroproline as a fire blight control option

The non‐protein amino acid 3,4‐dehydro‐l‐proline (DHP) significantly reduced the incidence of fire blight infection on immature pear fruits infected with wildtype Erwinia amylovora. DHP also inhibited biofilm formation in both streptomycin‐sensitive and ‐resistant strains of E. amylovora and induced dispersal of preformed biofilms in the streptomycin‐sensitive strain. The investigations shed light on the hitherto undiscovered ability of DHP to inhibit bacterial biofilms and its potential as a chemical control option for fire blight.

Erwinia amylovora expresses fast and simultaneously hrp/dsp virulence genes during flower infection on apple trees.

BackgroundPathogen entry through host blossoms is the predominant infection pathway of the Gram-negative bacterium Erwinia amylovora leading to manifestation of the disease fire blight. Like in other economically important plant pathogens, E. amylovora pathogenicity depends on a type III secretion system encoded by hrp genes. However, timing and transcriptional order of hrp gene expression during flower infections are unknown.Methodology/Principal FindingsUsing quantitative real-time PCR analyses, we addressed the questions of how fast, strong and uniform key hrp virulence genes and the effector dspA/E are expressed when bacteria enter flowers provided with the full defense mechanism of the apple plant. In non-invasive bacterial inoculations of apple flowers still attached to the tree, E. amylovora activated expression of key type III secretion genes in a narrow time window, mounting in a single expression peak of all investigated hrp/dspA/E genes around 24–48 h post inoculation (hpi). This single expression peak coincided with a single depression in the plant PR-1 expression at 24 hpi indicating transient manipulation of the salicylic acid pathway as one target of E. amylovora type III effectors. Expression of hrp/dspA/E genes was highly correlated to expression of the regulator hrpL and relative transcript abundances followed the ratio: hrpA>hrpN>hrpL>dspA/E. Acidic conditions (pH 4) in flower infections led to reduced virulence/effector gene expression without the typical expression peak observed under natural conditions (pH 7).Conclusion/SignificanceThe simultaneous expression of hrpL, hrpA, hrpN, and the effector dspA/E during early floral infection indicates that speed and immediate effector transmission is important for successful plant invasion. When this delicate balance is disturbed, e.g., by acidic pH during infection, virulence gene expression is reduced, thus partly explaining the efficacy of acidification in fire blight control on a molecular level.

Study Regarding Effectiveness of Some Plant Extracts and Different Pesticides against an Erwinia Amylovora (Burrill.) Winslow et al. Strain Isolated from Quince

Study Regarding Effectiveness of Some Plant Extracts and Different Pesticides against anErwinia Amylovora(Burrill.) Winslowet al.Strain Isolated from QuinceThe interest in management fire blight prevention and control increased considerably in last years due to pathogen spread almost throughout the country. The objective of this paper focuses on testing of some plant extracts such asSalvia officinalis, Thymus serpyllum, Pelargonium odoratissimum, Hedera helix, Ocimum basilicum, Levisticum officinale, Tagetes patula, Galium verumand different pesticides against anErwinia amylovorastrain isolated from quince dameged samples. In this study, the biological material was represented by quince shoots, harvested from pomological collection of "Vasile Adamachi" farm, which belongs to University of Agricultural Science and Veterinary Medicine Iaşi. The research were performed in the Phytopathology laboratory. Following the experiment performed note that among the eight plant extracts a good antibacterial activity hasPelargonium odoratissimum, Ocimum basilicumandSalvia officinalisand of the three pesticides, Alcupral 50 PU. Contrary to expectations worst results were obtained after testing Aliette 80 WG andHedera helixproducts. The results were estimated statisticaly, by performing ANOVA test.

Phage therapy for plant disease control with a focus on fire blight

Abstract The concept of using bacteriophages (bacterial viruses) as biocontrol agents in pest management emerged shortly after their discovery. Although research on phage-based biopesticides temporarily stopped with the advent of antibiotics, the appearance of antibiotic resistant bacterial strains led to a renewed interest in phage therapy for control of plant diseases. In the past twenty years numerous successful experiments have been reported on bacteriophage-based biocontrol measures, and several comprehensive studies have recently been published discussing detailed results of phage application practices in pest management, mainly from North American authors. The present review focuses on bacteriophage-mediated control of fire blight (caused by Erwinia amylovora (Burill) Winslow et al.), the most devastating bacterial disease of pome fruits. Research results from North America are discussed along with recent data from European laboratories.

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.

Silencing of flavanone-3-hydroxylase in apple (Malus × domestica Borkh.) leads to accumulation of flavanones, but not to reduced fire blight susceptibility

Transgenic antisense flavanone-3-hydroxylase apple plants were produced to mimic the effect of the agrochemical prohexadione-Ca on apple leaves. This enzyme inhibitor for 2-oxoglutarate dependent dioxygenases is used as a growth retardant and for control of secondary fire blight of leaves. Like using the agent, silencing of flavanone-3-hydroxylase leads to an accumulation of flavanones in leaves, but in contrast not to the formation of 3-deoxyflavonoids. In prohexadione-Ca treated leaves the 3-deoxyflavonoid luteoforol is formed from accumulating flavanones, acting as an antimicrobial compound against the fire blight pathogen Erwinia amylovora. Seemingly, the silencing of just one of the 2-oxoglutarate dependent dioxygenases (in apple also flavonol synthase and anthocyanidin synthase take part downstream in the pathway) does not provide a sufficiently high ratio of flavanones to dihydroflavonols. This seems to be needed to let the dihydroflavonol-4-reductase/flavanone-4-reductase enzyme reduce flavanones to luteoforol, and to let this be reduced by the leucoanthocyanidin-4-reductase/3-deoxyleucoanthocyanidin-4-reductase, each acting with their respective weak secondary activities. Accordingly, also the intended inducible resistance to fire blight by prohexadione-Ca is not observed with the antisense flavanone-3-hydroxylase apple plants. On the other hand, for most transgenic lines with strong flavanone-4-reductase down-regulation, up-regulation of gene expression for the other flavonoid genes was found. This provides further evidence for the feedback regulation of flavonoid gene expression having been previously reported for the prohexadione-Ca inhibited apple plants.

Antibiosis Activity of Pantoea agglomerans Biocontrol Strain E325 Against Erwinia amylovora on Apple Flower Stigmas

Pantoea agglomerans E325, the active ingredient in a commercial product for fire blight control, was previously shown in vitro to produce a unique alkaline- and phosphate-sensitive antibiotic specific to Erwinia amylovora. Antibiosis was evaluated as a mode of antagonism on flower stigmas using two antibiosis-deficient mutants. On King's medium B, mutants E325ad1 and E325ad2 have stable smooth-butyrous or hypermucoid colony morphologies, respectively, and the parental strain E325 exhibits phenotypic plasticity with predominantly hypermucoid colonies accompanied by slower-growing, smooth-butyrous colonies. Mutants were tested against E. amylovora on stigmas of detached flowers of crab apple (Malus mandshurica) in growth chambers and apple (Malus domestica) in the orchard. Epiphytic fitness of the antibiosis-negative mutants was similar or greater than the parental strain as determined by relative area under the population curve (RAUPC). In laboratory and orchard trials, both mutants had significantly lower inhibitory activity against the pathogen (i.e., less reduction of E. amylovora RAUPC) compared with the parental strain. E325 and the mutants caused similar decreases in pH in a broth medium, indicating that acidification, which was previously reported as a possible mechanism of pathogen inhibition on stigmas, is not directly related to antibiosis. In this study we provide the first evidence for E325 antibiosis involved in E. amylovora growth suppression on apple flower stigmas.

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

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. 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. 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. 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.

Molecular and physiological properties of bacteriophages from North America and Germany affecting the fire blight pathogen Erwinia amylovora.

SummaryFor possible control of fire blight affecting apple and pear trees, we characterized Erwinia amylovora phages from North America and Germany. The genome size determined by electron microscopy (EM) was confirmed by sequence data and major coat proteins were identified from gel bands by mass spectroscopy. By their morphology from EM data, φEa1h and φEa100 were assigned to the Podoviridae and φEa104 and φEa116 to the Myoviridae. Host ranges were essentially confined to E. amylovora, strains of the species Erwinia pyrifoliae, E. billingiae and even Pantoea stewartii were partially sensitive. The phages φEa1h and φEa100 were dependent on the amylovoran capsule of E. amylovora, φEa104 and φEa116 were not. The Myoviridae efficiently lysed their hosts and protected apple flowers significantly better than the Podoviridae against E. amylovora and should be preferred in biocontrol experiments. We have also isolated and partially characterized E. amylovora phages from apple orchards in Germany. They belong to the Podoviridae or Myoviridae with a host range similar to the phages isolated in North America. In EM measurements, the genome sizes of the Podoviridae were smaller than the genomes of the Myoviridae from North America and from Germany, which differed from each other in corresponding nucleotide sequences.

A rapid lateral-flow immunoassay for phytosanitary detection of Erwinia amylovora and on-site fire blight diagnosis

Fire blight is an invasive disease caused by Erwinia amylovora that threatens pome fruit production globally. Effective implementation of phytosanitary control measures depends upon rapid, reliable pathogen detection and disease diagnosis. We developed a lateral-flow immunoassay specific for E. amylovora with a detection limit of log 5.7 CFU/ml, typical of pathogen concentrations in symptomatic plant material. The simple assay had comparable sensitivity to standard culture plating, serum agglutination and nested PCR when validated for application in a phytosanitary laboratory as a confirmatory test of cultured isolates and for first-line diagnosis of phytosanitary samples that represent the full range of commercial, ornamental and forestry host species. On-site validation in ring-trials with local plant inspectors demonstrated robust and reliable detection (compared to subsequent plating and PCR analysis). The simplicity, inspector acceptance and facilitation of expedited diagnosis (from 2 days for laboratory submitted samples to 15 min with the immunoassay), offers a valuable tool for improved phytosanitary control of fire blight.

Multiple Treatment Meta-Analysis of Products Evaluated for Control of Fire Blight in the Eastern United States

The aim of this analysis was to estimate the effect sizes and consistency of products evaluated for fire blight control in the eastern United States over the last decade. Because only 3% of the 69 studies published from 2000 to 2008 explicitly presented a measure of within-study variability, a method for estimating the least significant difference (LSD) and, hence the sampling variance, for studies with at least two significant mean separations in the presented mean multiple comparisons was developed. Lin's concordance analysis indicated that the estimated LSD was an accurate predictor of the actual LSD based on 35 studies in a calibration evaluation (ρ(c) = 0.997). Separate multi-treatment random-effects meta-analyses were performed for three control categories: antibiotics, biological control, and plant defense-activating products and mean log response ratios relative to the nontreated controls ([Formula: see text]) were computed for each treatment and then back-transformed to obtain the mean percent disease control. None of the products evaluated performed as well as streptomycin, the standard product for fire blight control, for which the mean disease control was 68.6%. As a group, experimental antibiotics provided the best fire blight control with mean effect sizes ranging from 59.7 to 61.7%. Among the biological controls, the best control was noted for treatments combining the antibiotic streptomycin with a product based on Pantoea agglomerans (55.0% mean disease reduction) or Bacillus subtilis (53.9%). Mean disease control was 31.9, 25.7, and 22.6%, respectively, for products based on B. subtilis, Pantoea agglomerans, and Pseudomonas fluorescens without an antibiotic, suggesting that the higher efficacy of the combination treatments was due to the antibiotic. Among the plant defense-activating products, prohexadione calcium had the highest and most consistent effect size (50.7% control), while other products provided modest mean disease control of between 6.1 and 25.8%. Percent control values were significantly moderated by study location and cultivar used in the study, and were smaller, but more variable, when products were tested under high disease intensity compared with low disease intensity. Results indicate that wide-scale use of biological control and plant defense-activating products in the eastern United States is likely to remain low.

BIOLOGICAL CONTROL AGENTS OF FIRE BLIGHT: SUCCESSES AND CHALLENGES

Because of the limited number of products available for control of fire blight, the search for alternative methods of control has been a dynamic and productive field of scientific research. Today, only a few biological control agents are registered for control of this disease, representing a relatively small part of the market for products to control fire blight. This is in spite of fire blight being an economically important bacterial disease whose impact could be significantly lowered by simply protecting the flowers from the pathogen, a task that many biological control agents seem able to do. The number of new biological control agents being commercialised might be restricted by problems linked to the registration process. This is especially true for biological control agents that belong to species containing poorly characterised strains of medical or environmental interest. Furthermore, the biological control agents available in the USA were reported to exhibit a much lower and more variable efficacy than streptomycin. This may contribute to the choice by growers to use antibiotics more often than they use biological control agents. The future of biological control agents for the control of fire blight might rest in the ability to incorporate such products in new strategies of control. These strategies might include the use of several biological control agents, but may also include antibiotics or some elicitors.

KASUMIN: FIELD RESULTS FOR FIRE BLIGHT MANAGEMENT AND EVALUATION OF THE POTENTIAL FOR SPONTANEOUS RESISTANCE DEVELOPMENT IN ERWINIA AMYLOVORA

The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L (Arysta Corp.; Cary, NC) for control of fire blight was evaluated in six experiments conducted over four field seasons in our experimental orchards in East Lansing, MI. Blossom blight control was statistically equivalent to the industry standard streptomycin in all experiments. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to 250 or 500-ppm Ks was not observed when cells were directly plated on medium containing high concentrations of the antibiotic. However, exposure to increasing concentrations of Ks in media (initial conc. 25 µg ml-1) resulted in the selection of Ks resistance in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated they harbored mutations in the kasugamycin target ksgA gene and that all mutants were impacted in relative fitness observable through a reduced growth rate in vitro and decreased virulence in immature pears. Our data confirm the importance of kasugamycin as an alternate antibiotic for fire blight management and lay the groundwork for the development and incorporation of resistance management strategies.

Improvement of fitness and efficacy of a fire blight biocontrol agent via nutritional enhancement combined with osmoadaptation.

The efficacy of Pseudomonas fluorescens EPS62e in the biocontrol of Erwinia amylovora was improved by a procedure of physiological adaptation to increase colonization and survival in the phytosphere of rosaceous plants. The procedure consisted of osmoadaptation (OA) and nutritional enhancement (NE). OA was based on saline stress and osmolyte amendment of the growth medium during inoculum preparation. NE consisted of addition of glycine and Tween 80 to the formulation. NE and OA increased the growth rate and carrying capacity of EPS62e under high-relative-humidity (RH) conditions and improved survival at low RH on flowers under controlled environmental conditions. NE did not promote growth or affect infection capacity of E. amylovora. The effect of both methods was tested in the field by following the population of EPS62e using quantitative PCR (Q-PCR) (total population) and CFU counting (culturable population) methods. Following field application, EPS62e colonized blossoms, but it was stressed, as indicated by a sharp decrease in culturable compared to total population levels. However, once established in flowers and at the end of bloom, almost all the total population was culturable. The physiological adaptation treatments increased population levels of EPS62e over those of nonadapted cells during the late stage of the flowering period. Control of fire blight infections in flowers and immature fruits was tested by field application of EPS62e and subsequent inoculation with E. amylovora under controlled-environment conditions. The efficacy of fire blight control increased significantly with the combination of nutritional enhancement and osmoadaptation, in comparison with the absence of physiological adaptation.