Crab apple blossoms as a model for research on biological control of fire blight

ABSTRACT Nonseasonal availability of pomaceous flowers could improve laboratory detection and prefield testing of biocontrol agents for fire blight of pear and apple. Crab apple was selected as a model because of its high flower productivity on 1-year-old wood, high susceptibility to fire blight, and availability from nurseries. Cultivars Manchurian and Snowdrift were manipulated to bloom once by transferring dormant nursery trees from a cold room to a greenhouse and a second time by defoliating trees and applying 1% cytokinin and 0.1% gibberellins to the buds with a brush. Different sets of trees were induced at different times to bloom, so that flowers were produced 12 months in the year. When known bacterial antagonists (Erwinia herbicola strain C9-1 and Pseudomonas fluorescens strain A506) were applied alone or in combination to the stigmas of detached crab apple blossoms prior to inoculation with the pathogen (E. amylovora strain Ea153), population interactions over time were comparable to those reported in previous studies involving pear or apple. In a subsequent series of experiments, the relative effects of 12 bacterial strains on stigmatic populations of strain Ea153 were similar for detached blossoms of crab apple in the laboratory, blossoms of intact crab apple trees in the greenhouse, and blossoms of pear and apple in the field. Additionally, when stigmas of detached crab apple blossoms were inoculated with antagonists (strains C9-1 and A506) and the pathogen, and later subjected to a 24-h wetting period, bacterial populations in the flower hypanthium increased and disease was suppressed. These studies indicate that crab apple blossoms can serve as a suitable model for year-round evaluation and study of biocontrol agents 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.     

Potential and limits of acylcyclohexanediones for the control of blossom blight in apple and pear caused by Erwinia amylovora

Growth-regulating acylcyclohexanediones such as prohexadione-calcium and trinexapac-ethyl have been shown to be effective in controlling fire blight infections on shoots. Since blossoms represent the primary site of infection for the fire blight pathogen, Erwinia amylovora , trinexapac-ethyl and prohexadione-calcium were evaluated for their ability to reduce fire blight infection on apple and pear flowers. Field experiments and experiments under controlled conditions were conducted on apple flowers for 4 years. A reduction of up to 50% of blossom blight was observed in treated plants. In addition, treatment with trinexapac-ethyl reduced up to the 77% the percentage of fireblight-affected flowers from which disease progressed into shoots. On pear, numbers of flower infections were reduced by a quarter and flower infections leading to diseased shoots was reduced by up to 50%. Mechanisms underlying diseased reduction following treatment with the two acylcyclohexanediones was studied using a confocal laser scanning microscope combined with a gpf -labelled strain of E. amylovora . These non-invasive techniques demonstrated bacterial migration was reduced by up to 60 and 66% in apple and pear xylem, respectively.     

Stem injection of prohexadione carboxylic acid to protect blossoms of apple trees from fire blight infection (Erwinia amylovora)

Journal of Plant Diseases and Protection - Stem injection of prohexadione carboxylic acid (pca) was tested to prevent blossom infection by the fire blight pathogen Erwinia amylovora on apple trees,...     

Biological control of fire blight of hawthorn (Crataegus monogyna) with Erwinia herbicola under protected conditions

Isolates of Erwinia herbicola , obtained from flowers and leaves of hawthorn ( Crataegus monogyna) , were screened as potential control agents of fire blight disease (caused by Erwinia amylovora) using an immature pear fruit assay. Selected isolates were subsequently tested for disease control by infection of hawthorn blossom in the laboratory, and by shoot infection of hawthorn plants grown under controlled (glasshouse) and fluctuating (polythene tunnel) environmental conditions.
Although the immature pear fruit assay provided a general screen for the selection of antagonists for the control of both blossom and shoot blight, it had two major limitations when quantitatively applied. Firstly there were inconsistencies in the relative effects of different isolates on the pear-slice surface, with some isolates being more suppressive than the standard antagonist Eh252 in the first screening and less in the second. Secondly the assay was not able to predict accurately the level of control in the intact plant-as no correlation occurred between the level of control in the pear fruit assay and the percentage control of either blossom blight or shoot blight.
Two isolates of E. herbicola , WL9 and WL40, reduced both blossom- and shoot-blight. WL9 provided over 80% control of blossom blight, equivalent to that provided by chemical agents, and also gave total control of shoot blight when applied at a WL9: pathogen ratio of 10:1.     

The various means of dissemination of the fire blight bacterium Erwinia amylovora

A brief review is presented of the means of dissemination of Erwinia amylovora. The bacterium exists in the form of ooze, strands, epiphytic populations and endophytic populations. The status of endophytic bacteria in budwood and fruits is discussed. International dissemination of fire blight has taken place by wind, probably by birds, and by movement of budwood. It has been suggested that fruits may be involved, but there is international disagreement whether measures should be taken to protect against this pathway of dissemination.     

Novel metrics to classify fire blight resistance of 94 apple cultivars

Fire blight (Erwinia amylovora), a potentially devastating disease in apple, can cause floral, fruit and structural damage and even tree death. Most commercial apple cultivars are susceptible and the resistance/susceptibility of many modern cultivars has not been evaluated. Fire blight resistance/susceptibility is difficult to phenotype due to quantitative resistance, impacts of tree vigour and environment on susceptibility, and the erratic nature of the disease. Resistance/susceptibility levels were determined for 94 apple cultivars and important breeding parents. In 2016 and 2017, multiple actively growing shoots per tree (about three trees per cultivar) were challenged with E. amylovora Ea153n via a cut-leaf inoculation method. Proportion of current season's shoot length blighted (SLB) was calculated for each shoot. To classify cultivar responses, estimated marginal SLB means were compared to four controls, representing highly susceptible (HS) to highly resistant (HR), via Dunnett's tests. Cultivar responses ranged from HS to HR with estimated marginal SLB means of 0.001–0.995 in 2016 and 0.000–0.885 in 2017. Most cultivars demonstrated similar resistance/susceptibility levels in both years (ρ = 0.657, P < 0.0001). K-means clustering was used to classify cultivars into three resistance/susceptibility groups based on incidence, average severity (SLB), and maximum severity values (maximum SLB and age of wood infected). Sixteen cultivars were consistently moderately resistant (MR) to HR while the remainder ranged from HS to MR. An updated comparison of susceptibility of important cultivars is provided. Resistance/susceptibility information gained could be used to identify genetic loci associated with resistance/susceptibility and/or inform parental selection in apple scion breeding programmes.     

Transfer from Erwinia herbicola to Escherichia coli of a plasmid associated with biocontrol of fire blight

Strains of Erwinia herbicola effective in the biocontrol of fire blight of hawthorn were used to investigate the possibility that the antagonistic activity is coded by plasmid-born genes. Agarose gel electrophoresis of isolated plasmids from four antagonistic Erw. herbicola strains showed a band of a supercoiled 12 kb plasmid in each strain, with a second band greater than 16.2 kb consistently seen in two strains. Erw. herbicola strains showed resistance to penicillin-G, which could be conferred on penicillin-G sensitive Escherichia coli TG1 by transformation with a pure Erw. herbicola plasmid preparation. Transformed strains of Esc. coli appeared to contain the Erw. herbicola 12 kb plasmid, but not the > 16.2 kb plasmid. In an agar plate assay, Esc. coli transformants produced an inhibition zone against Erw. amylovora similar to those produced by the original Erw. herbicola strains. In two biocontrol assays, the transformed Esc. coli strains had a suppressive effect on disease development on infected pear fruit slices and hawthorn blossoms.     

梨火疫病菌对噻唑锌及其他几种杀细菌剂的敏感性

噻唑锌是近期在我国登记为防治梨火疫病的新型杀菌剂。为明确梨火疫病菌Erwinia amylovora对噻唑锌及其他几种杀细菌剂的敏感性现状,本研究选用2016年-2020年从新疆不同地区分离的50个E. amylovora 代表菌株,通过药剂-病原菌共培养结合平板菌落法测定E. amylovora 对噻唑锌的敏感性,比较不同来源菌株的敏感性差异,并分析E. amylovora 对噻唑锌与农用链霉素、噻霉酮和春雷霉素的敏感性差异。结果表明,来自新疆不同地区、不同寄主和不同年份的E. amylovora 菌株对噻唑锌的敏感性差异明显,EC50分布在2.67~38.13 μg/mL, 平均（18.60±6.35） μg/mL。阿克苏地区阿瓦提县的杜梨分离菌株H10最敏感,EC50为2.67 μg/mL,巴州轮台县的山楂分离菌株Y126的敏感性最低,EC50为38.13 μg/mL,二者的EC50相差13.28倍。供试E. amylovora 菌株对噻唑锌的敏感性频率分布呈连续的单峰曲线,符合正态分布,将EC50平均值(18.60±6.35)μg/mL确定为新疆梨火疫病菌对噻唑锌的敏感基线,适用于新疆梨火疫病菌对噻唑锌的抗药性检测。供试E. amylovora 菌株对噻唑锌的抗性水平为0.13~2.05 (均值为1.00±0.35),均为敏感菌株,未发现抗药性菌株。相关性分析表明,E. amylovora 对噻唑锌的敏感性与农用链霉素（r=0.138,P=1.56）、噻霉酮(r=0.417,P=0.78)和春雷霉素(r=0.434,P=0.65)的敏感性之间不存在相关性(P>0.05)。本研究结果为后期评估梨火疫病菌对噻唑锌的抗药性风险奠定了基础,为制定梨火疫病的合理用药策略提供了依据。     

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.     

Induction of resistance to fire blight in apple by acibenzolar-S-methyl and DL-3-aminobutyric acid

Journal of Plant Diseases and Protection - Fire blight disease caused by Erwinia amylovora is one of the most important bacterial diseases of apple and pear world-wide. DL-3-aminobutyric acid...     

梨火疫病菌的RPA快速检测技术的研发

梨火疫病菌Erwinia amylovora是我国进境植物检疫性有害生物, 主要侵染梨、苹果、山楂、海棠等多种蔷薇科植物引起梨火疫病。该病自1780年在美国被发现以来, 已经传播到世界多个国家, 对我国的果树生产也构成一定的威胁。严格植物检疫是全球防控梨火疫病菌扩散危害的重要方法之一, 目前我国植物检疫部门主要从实验室分离鉴定、分子检测和田间症状识别及快速检测方面开展工作。本研究基于重组酶聚合酶扩增(RPA)技术和E.amylovora 16S-23S ITS区间的保守序列设计的引物和探针, 建立了实时荧光RPA和侧流层析试纸条RPA两种检测方法。经测试, 在39℃条件下, 这两种检测方法都具有良好的特异性, 分别能在20 min和10 min内完成扩增。实时荧光RPA对E. amylovora菌株DNA的检测阈值为1.38×10-2 ng/μL;侧流层析试纸条RPA对E. amylovora菌悬液和DNA的检测阈值分别为104 cfu/mL和1.38×10-3 ng/μL。侧流层析试纸条RPA体系因反应快速、读取结果方便、不需要复杂仪器操作可以满足现场快速检测的需求。     

梨火疫细菌实时荧光PCR和诱捕PCR-ELISA检测方法的建立

根据梨火疫细菌中独特而保守存在的质粒pEA29，设计了1对引物和3条探针，建立了实时荧光PCR检测方法和诱捕PCR-ELISA检测方法。实时荧光PCR采用带荧光标记的核酸杂交探针，边扩增边检测，步骤简单，不需PCR后处理，可避免假阳性和交叉污染；诱捕PCR-ELISA检测方法只需简单处理的样品就能检测，减少了核酸不纯出现的漏检，由于增加了核酸杂交探针，可不需凝胶电泳EB染色检测，不会出现假阳性问题。     

Notes on the role of water potential in the pathogenesis of fire blight,caused by Erwinia amylovora

The rate of multiplication of fire blight causing bacteriumErwinia amylovora (Burrill) Winslow et al. depends on the availability of water. Water availability can be quantified by means of the parameter water potential. The relationship between water potential and relative multiplication rate ofE. amylovora was derived from experiments of L. Shaw (1935). This relationship appears to be applicable toE. amylovora in plant tissues and in nectar of flowers.Multiplication and expansion ofE. amylovora in a restricted space, e.g. an intercellular hole, creates a pressure, which may cause schizogenic cavities in soft tissue. Strong tissue, however, may be able to resist this multiplication pressure of the bacteria, so that symptom progression can be prevented. A hypothesis is formulated on how the multiplication pressure may be quantified by means of the parameter water potential. Expansion of bacterial ooze may alo be due to absorbtion of water without increase of dry weight (e.g. a daily cycle of shrinkage and expansion). This expansion may give rise to a swelling pressure, which again may be quantified by means of the parameter water potential.Samenvatting De vermenigvuldigingssnelheid van de bacterie die bacterievuur veroorzaakt (Erwinia amylovora (Burrill) Winslow et al.,), hangt af van de beschikbaarheid van water. De beschikbaarheid van water kan worden gekwantificeerd met de parameter waterpotentiaal. De relatie tussen waterpotentiaal en relatieve vermenigvuldigingssnelheid vanE. amylovora werd afgeleid uit experimenten van L. Shaw (1935, Cornell University Agricultural Experiment Station, Ithaca. Memoir 181). Deze relatie kan zowel worden toegepast op de pathogenese in planteweefsel als op de epifytische ontwikkeling in nektar.Vermenigvuldiging vanE. amylovora in een beperkte ruimte, bijvoorbeeld in een intercellulaire holte, creëert een druk, die tot scheuren van zacht weefsel kan leiden. Sterk weefsel kan de vermenigvuldigingsdruk van de bacteriën vermoedelijk wel weerstaan, zodat uitbreiding wordt verhinderd. In een hypothese wordt beschreven hoe de vermenigvuldigingsdruk zou kunnen worden gekwantificeerd met behulp van de parameter waterpotentiaal. Wateropname door bacterieslijm zonder toename van het drooggewicht (bijvoorbeeld een dagelijkse gang van krimpen en zwellen) kan ook leiden tot een druk. Deze druk kan eveneens worden berekend met de parameter waterpotentiaal.     

Real‐time PCR detection of Erwinia amylovora on blossoms correlates with subsequent fire blight incidence

Fire blight is the most devastating bacterial disease of rosaceous plants. Forecasting fire blight infections is important to allow for countermeasures that reduce economic damage in pome fruit production. Current computerized forecasting models are solely based on physical factors such as temperature and moisture, but not on the actual presence of the pathogen Erwinia amylovora. Although the inoculum concentration is considered to be crucial for infection and disease outbreak, most current approaches used for identification of fire blight inoculum including morphological, biochemical, serological, and DNA‐based methods are nonquantitative. Based on a real‐time PCR approach previously published, an improved protocol to be used directly on whole bacteria in the field is described. The method allows for early detection and quantification of the pathogen prior to the occurrence of first symptoms. There is a clear correlation between bacterial abundance and subsequent disease development. However, in some cases, no disease symptoms could be observed despite a pathogen load of up to 3·4 × 10⁶ cells per blossom. Integration of the amount of pathogen detected into refined prediction algorithms may allow for the improvement of applied forecasting models, finally permitting a better abatement of fire blight.     

Effects of pear tree physiology on fire blight progression in perennial branches and on expression of pathogenicity genes in Erwinia amylovora

The interaction between Erwinia amylovora (the causal agent of fire blight) and the physiological status of pear trees was examined under orchard conditions. The physiological status of the trees was defined qualitatively, using host phenology and vigour as measures, and quantitatively, using the sorbitol content in annual shoots as a measure. Qualitatively, tree response to fire blight was governed by phenological stage at the time of infection and vigour: low vigour trees inoculated in the autumn (just before entering dormancy) and high vigour trees inoculated in the spring (soon after bloom) were more susceptible than high vigour trees inoculated in the autumn and low vigour trees inoculated in the spring. Quantitatively, the rate of symptom progression in perennial branches (SPR) was significantly (P ≤ 0.001) correlated to the absolute value of the rate of sorbitol content change (|SCR|). The relationship between hrp genes expression of transformed E. amylovora (estimated according to hrpE and hrpJ expression) and |SCR| was determined on 1 year-old trees. Expression of hrp genes was significantly correlated with |SCR| (P = 0.004) and 63.5% of the variability in the hrp genes expression was attributed to |SCR| values. The expression of hrp genes increased gradually and asymptotically with increasing |SCR| values; further increase in |SCR| did not affect the expression.     

Correction to: Phenotypic and marker-assisted characterization of new apple genotypes with high resistance to fire blight

European Journal of Plant Pathology - A Correction to this paper has been published: https://doi.org/10.1007/s10658-021-02315-7     

Evaluation of kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of kasugamycin resistance potential in Erwinia amylovora

The emergence and spread of streptomycin-resistant strains of Erwinia amylovora in Michigan has necessitated the evaluation of new compounds effective for fire blight control. The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L 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. E. amylovora populations remained constant on apple flower stigmas pretreated with Kasumin and were ≈100-fold lower than on stigmas treated with water. Kasumin applied to apple trees in the field also resulted in a 100-fold reduced total culturable bacterial population compared with trees treated with water. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development and acquisition of a transferrable Ks(R) gene. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to Ks at 250 or 500 ppm 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 concentration 25 μg ml(-1)) resulted in the selection of Ks resistance (at 150 μg ml(-1)) in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated that 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 pear fruit. The possible occurrence of a reservoir of Ks(R) genes in orchard environments was also examined. Culturable gram-negative bacteria were surveyed from six experimental apple orchards that had received at least one Kasumin application. In total, 401 Ks(R) isolates (42 different species) were recovered from apple flowers and leaves and orchard soil samples. Although we have not established the presence of a transferrable Ks(R) gene in orchard bacteria, the frequency, number of species, and presence of Ks(R) enterobacterial species in orchard samples suggests the possible role of nontarget bacteria in the future transfer of a Ks(R) gene to E. amylovora. 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.