Real-time PCR for detection and quantification of Erwinia amylovora, the causal agent of fireblight

Real-time PCR was used for quantitative detection of Erwinia amylovora , the causative agent of fireblight. Specific primers were created from a DNA fragment of the common plasmid pEA29, successfully used for standard PCR identification of the pathogen. The primers amplified DNA from various E. amylovora strains, but not from other plant-associated bacteria. DNA of E. amylovora was also amplified from field samples and from inoculated apple leaves or flowers. Neither the presence of other bacteria nor low amounts of tissue extracts from bark or leaves changed the signal threshold. Assays with SYBR Green I instead of the Taqman probe showed a similar sensitivity, detecting 50 cells per assay. Real-time PCR could be especially useful for mass screening of commercial products and for resistance studies of transgenic host plants, in breeding experiments and after treatments to control fireblight.     

Fireblight in Israel—1985. Observations and recommendations

Fireblight, caused by the bacterium Erwinia amylovora , was observed in Israel for the first time in 1985. Primarily, the disease affected several pear orchards. The pathogen was also isolated and identified from symptomatic tissue of apple and cotoneaster. The occurrence, severity and distribution of the disease in 1985 is discussed in relation to environmental conditions and horticultural practices. Although fireblight was widespread in 1985, overall damage from the disease was limited. Actions taken to confirm the presence of fireblight and to manage the disease during its first season of occurrence are discussed. Prospects for the occurrence of fireblight in Israel in the future are discussed and specific recommendations for management of the disease are presented. Based on its behaviour in other countries, fireblight is expected to become established in Israel and the disease will require constant management to minimize losses.     

Occurrence of Erwinia amylovora on pear in Albania

Pear trees were observed in the province of Durres (AL) with typical symptoms of fireblight on the shoots, fruit and branches. Bacteriological analysis of samples of diseased material confirmed the presence of Erwinia amylovora. The isolates were identified on the basis of phenotypical and pathogenic characteristics. The total protein electrophoretic profiles of the Albanian isolates were indistinguishable from those of the strain found in the Po valley (IT).     

Fireblight at Long Ashton: a case history, 1971–1983

At Long Ashton Research Station serious outbreaks of fireblight (caused by Erwinia amylovora ) occurred on apples in 1978, 1980 and 1982. and on pears in 1979, Mid-season and late-flowering cider apple cultivars were particularly affected. On cider apple, outbreaks were associated with years of profuse blossom coinciding with unusually warm temperatures during bloom. Spread of infection between hosts, including hawthorn and cotoneaster, was undoubtedly important. These and other factors underlying the outbreaks are discussed.     

First occurrence of fireblight in Romania

Fireblight was first observed in Romania in 1992-08 near Braila, and shortly after in the pear collection of the Fruit Research Institute in Pitesti-Maracineni. Advanced symptoms indicated that the disease was present at least one year earlier. Symptomatology, fruit pathogenicity, selective culture media, hypersensitivity, agglutination and fatty acid class analysis confirmed the authenticity of the pathogen as Erwinia amylovora. Of the native Romanian fruit cultivars affected, apple cv. Delicios de Voinesti and pear cv. Aromat de Bistrita appear the most susceptible to fireblight.     

Alternative methods to describe virulence of Erwinia amylovora and host-plant resistance against fireblight

The evaluation of host-plant susceptibility to Erwinia amylovora and of colonization of host-plant tissue by individual strains was facilitated by labelling the pathogen with green fluorescent protein (GFP). Colonization of apple leaves assayed with a fluorescence microscope was associated with visual disease ratings on plants to describe virulence (= aggressiveness) of the fireblight pathogen. Resistance induced with 2,6-dichloro-isonicotinic acid (INA) and benzo(1,2,3-) thiadiazol-7-carbothioic acid-S-methyl ester (BTH, the active component of BION™) restricted colonization by the pathogen to an area adjacent to the inoculation site. Migration in leaves was associated with symptom formation on pear slices and host plants of mutant strains. Non-virulent E. amylovora mutants did not migrate into the leaf veins and strains with intermediate-to-low virulence moved slowly. To compare the migration efficiency of individual wild-type strains in apple and plum cultivars, a blend of five wild-type E. amylovora strains with specific numbers of short-sequence DNA repeats (SSRs) in the common plasmid pEA29 was applied to distinguish them by PCR. Fast-moving strains identified in the GFP assays were dominant, independent of the apple cultivar. When apple shoots, pear slices or leaves of apple plants were coinoculated with streptomycin (Sm)-resistant strains and the corresponding parent strains, Sm-resistant mutants were able to dominate the wild-type strain for tissue colonization.     

Survival of Erwinia amylovora on pears and on fruit containers in cold storage and outdoors

The survival of Erwinia amylovora during cold storage or outdoors may be a relevant factor in the spread of fireblight. The survival of E. amylovora was studied in cold storage on pear fruits, on container materials and on packaging paper, and outdoors on wood (oak and poplar) and on polyethylene. The samples were contaminated with a bacterial suspension of a mutant strain, washed, concentrated by centrifugation, and the final concentrates were used for plate counting. In cold storage, reisolation from the calyx was successful even after 101 days, whereas on pear surfaces, it was unsuccessful after just 1 day. On oak and poplar wood, reisolation was obtained up to 77 days in cold storage for both types of wood, but only up to 27 and 55 days, respectively, outdoors. Reisolation from packaging paper in cold storage was successful up to 14 days. Reisolation from polyethylene outdoors was unsuccessful after 24 h. Survival curves were calculated for each material. On the basis of a model of inoculum transmission, and using the survival curves, a phytosanitary risk period for the different types of materials was estimated.     

Effectiveness of the resistance inducer prohexadione‐Ca against fireblight in shoots of apple trees inoculated with Erwinia amylovora1

Due to the lack of effective, non‐phytotoxic and publicly acceptable materials for controlling fireblight in pome fruit trees, novel strategies against Erwinia amylovora are being sought. Resistance‐inducing compounds, such as prohexadione‐Ca, represent promising alternatives. Prohexadione‐Ca is the active substance of the bioregulator Regalis, currently being introduced in several European countries and overseas. Prohexadione‐Ca reduces shoot elongation due to inhibition of gibberellin biosynthesis. Furthermore, it leads to significant changes in the spectrum of flavonoids and their phenolic precursors in pome fruits, which causes reduced susceptibility to fireblight and other pathogens. In 2002 and 2003, container‐grown apple trees of the cultivars ‘Idared’ and ‘Freedom’ were treated with different dosages of prohexadione‐Ca two weeks before inoculation with E. amylovora. The effect of prohexadione‐Ca against shoot blight was determined by measuring the lengths of necrotic lesions and symptoms on vascular bundles caused by the pathogen. Treatments with prohexadione‐Ca turned out to be much superior to the ones with streptomycin, kasugamycin and a bacterial antagonist, which were used for comparison. Acibenzolar‐S‐methyl (Bion), another resistance‐inducing compound, was included in some of the experiments and gave intermediate results. The simultaneous control of excessive shoot growth and shoot infections by fireblight is seen as a major advantage of using prohexadione‐Ca in pome fruit trees.     

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.     

Fireblight (Erwinia amylovora) of pome fruits in Hungary: national phytosanitary measures and management of the disease

After the first detection of Erwinia amylovora in Hungary, as a result of an intensive survey started in spring 1996, it was concluded that the incidence of fireblight was highest in a zone 10–30 km wide and 200–250 km long near the southern and south-eastern border. The disease was concentrated in the Békés, Bács-Kiskun and Csongrád counties. Isolated infection foci were, however, also found in Baranya county. In 1997, further spread of the disease was recorded. Only three of the 19 counties could be considered as free from the disease. The disease attacked nine major host plants, but the majority of infected plants were quince and pear. During the eradication campaign in 1996, more than 60 000 trees were uprooted and destroyed across the country. Eradication was performed partly by special work teams and partly with the participation of growers. The pathogen most probably entered Hungary by aerial drift from Romania or Yugoslavia during the previous 2 or 3 years. In the interior of the country, introduction through nursery stock and possibly infested tools, as well as wind and insect movement, cannot be excluded. Major measures to control the disease are discussed.     

Evaluation of Likelihood of Co-Occurrence of Erwinia amylovora with Mature Fruit of Winter Pear

ABSTRACT Phytosanitary concerns about fire blight prohibit export of U.S.-grown pears to some countries without this disease. To examine these concerns, we evaluated the potential for co-occurrence of Erwinia amylovora with mature, symptomless winter pear fruit by inoculation experiments and by survey of commercial orchards. Immature pear and apple fruit were inoculated in orchards with E. amylovora strain 153N as resuspended lyophilized cells or as ooze from diseased tissues. Regardless of inoculum source, population size of Ea153N on fruit declined by an order of magnitude every 3 to 4 days during the first 2 weeks after inoculation; at 56 days after inoculation, Ea153N was not detected, except on 1 of 450 fruit with 4 colony forming units (CFU). After inoculation of flowers, calyx-end survival of Ea153N on pear and apple fruit declined from high populations at petal fall to a few cells at harvest, with no detection of the pathogen after a 7-week cold storage. Migration of Ea153N into symptomless pear fruit from diseased branches was evaluated by enrichment assay and nested polymerase chain reaction of internal fruit core tissues; these assays failed to detect the pathogen in healthy fruit from diseased trees. At harvest, E. amylovora could not be detected on 5,599 of 5,600 fruit of d'Anjou pear sampled from commercial orchards in major production areas of the Pacific Northwest; one fruit yielded 32 CFU of the pathogen. Postharvest, mature pear fruit contaminated with Ea153N and subsequently wounded required a dose of >10,000 cells at the wound site to allow for persistence of the pathogen through a 7-week-cold storage. We conclude that epiphytic E. amylovora shows similar survival characteristics on both pear and apple fruit, this pathogen is not an endophyte within mature symptomless pear fruit, its presence is exceptionally rare on commercially produced fruit, and that epiphytic survival of E. amylovora through a postharvest chilling period is unlikely given the unrealistically high population size required for persistence.     

Using weather records and available models to predict the severity of fireblight should it enter and establish in Australia1

Australia is free from fireblight, a serious apple and pear disease. Published models and weather data from all major apple and pear-producing areas of Australia were used to predict the possible severity of the disease and losses in production should the disease enter and establish in Australia. The results suggest that fireblight could be severe in most areas in most seasons. In the worst case, with every area affected, this could result in up to 20% losses in apple production and up to 50% losses in pear production.     

Fireblight Inoculum: Sources and Dissemination1

Understanding the disease cycle of fireblight requires knowledge of the sources of inoculum and means of dissemination. In regions where the disease is endemic, hold–over cankers are the most important source of primary inoculum. Dissemination of primary inoculum occurs by rain or by crawling or flying insects. Often, blossoms infested with Erwinia atnylovora (Burrill) Winslow et al. provide secondary inoculum that is disseminated by pollinating insects or by rain. Later in the growing season, secondary inoculum produced in infected tissues may be disseminated by rain or by sucking and biting insects. Relatively rare sources of inoculum and inefficient means of dissemination may be responsible for introducing the pathogen to areas where fireblight has not been found previously. E. amylovora may be carried to new locations in intimate association with host tissues (infected nursery stock), in association with vectors (insects, birds or man), or by wind as “strands” or in airborne water drops. Every effort should be made to prevent introduction of E. amylovora into areas now thought to be free of fireblight. Attempts to eradicate the disease from several new areas in northern Europe have been unsuccessful. Because weather conditions and the host plants present in southern Europe are especially conducive to severe damage from fireblight, plant protection personnel are advised to prepare now for the possible introduction of disease. Personnel should be trained to recognize the disease and plans should be drawn to contain it or to manage host plants to minimize disease losses.     

Recent Progress in Breeding for Fireblight Resistance in Apples and Pears in North America1

Resistance in apple is evaluated by needle–inoculation of succulent shoot tips with 10⁶–10⁷ cells of a virulent isolate of Erwinia amylovora (Burr.) Winslow et al. (the incitant of fireblight) and measurement of the resulting cortical lesion when extension is complete. Data are now available on practically all commercial cultivars, some of which have a useful level of resistance. Some newer cultivars, particularly those with resistance to scab (Venluria inaequalis [Cooke] Wint.) derived from Malus floribunda, have good resistance to E. amylovora. A very high level of resistance is present in Asiatic Malus species, including M. x robusta, M. x sublo–bata, M. x atrosanguinea, and M. prunifolia, and in the North American species M. fusca. This type of resistance appears to be inherited polygenically, and because of its detectability in young seedlings can be used conveniently in breeding. Objectives of pear breeding programs are aimed at developing superior fruit quality combined with resistance to fireblight, psylla, and Fabraea leaf spot. Many high quality cultivars of Pyrus communis are extremely susceptible to E. amylovora and sensitivity appears to be controlled by a dominant gene Se. A high level of resistance is present in P. ussuriensis but varies considerably between clonal selections of other Pyrus species. Pear seedlings from controlled pollinations are artificially inoculated in the glasshouse with a similar bacterial suspension as used for apples, and only the most resistant ones are selected and planted in the field for future evaluation. In Beltsville, heritability studies of crosses between non–sensitive parents have indicated that selection for resistance within progenies results in a high degree of genetic gain. Interspecific hybridization has an advantage over P. communis crosses mainly when insect or fungus resistance is required.     

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.     

Official survey,in 2002, for detection of Erwinia amylovora in a proposed protected zone in Jihomoravsky region (Czech Republic)1

Inspectors of the Czech NPPO surveyed the occurrence of fireblight Erwinia amylovora in an area of Jihomoravsky region (South Moravia) proposed as an EU protected zone, including 16 designated buffer zones around nurseries. The disease was not detected in communes where fireblight hosts are grown (nurseries, variety testing stations, orchards) or in buffer zones around nurseries. In 902 communes where fireblight hosts are grown only in orchards or not at all, wild host plants were inspected at 2.629 observation points (2137 located by GARMIN GPS). In Vy?kov district, suspected fireblight was confirmed at one observation point on Crataegus, growing by a railway in Rousínov commune and, in the course of a delimiting survey outside observation points, in four other communes (Drnovice, Habrovany, Komo?any and Vy?kov).     

Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction

Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross-recognized by the production of a weaker PCR band of similar size to E. amylovora . In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 10² cfu mL^–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora -spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora , the PCR detection sensitivity was determined to be 6.6 × 10² cfu mL^–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.     

梨火疫病的分布,传播及检测技术研究进展

本文介绍了梨火疫病的危害,传播及目前世界分布;讨论了各种植物材料和包装物传病的危害性,评价了包括传统的和分子生物学方法在内的梨火疫病菌的各种检测方法及该病对我国的潜在威胁,为对该病的检疫检测提供理论依据。     

梨园气溶胶中梨火疫病菌的定量检测

为系统研究梨园气溶胶中梨火疫病菌的含量, 本研究于2019年－2021年在新疆库尔勒市人和农场梨园, 利用病原菌孢子捕捉器在每年春季(4月下旬)、夏季(6月中旬)、秋季(9月中旬)收集梨园气溶胶, 检测梨火疫病菌。结果显示, 健康梨园气溶胶中未检测到梨火疫病菌, 不同发病程度的梨园气溶胶中均能检测到梨火疫病菌, 携菌量均值在102 cfu/(24cm2·h)以上, 其中, 气溶胶中梨火疫病菌含量最高值为2.81×104 cfu/(24cm2·h), 最低值为8.50×102 cfu/(24cm2·h); 重度、中度、轻度发病果园收集的气溶胶中含梨火疫病菌总菌落数均值分别为8.74×103、4.55×103、2.36×103 cfu/(24cm2·h)。此外, 在同一高度收集的气溶胶中, 梨火疫病菌菌落数随收集时间的延长而增加。不同季节气溶胶携菌量检测结果表明, 秋季发病梨园中气溶胶携菌量明显高于夏季和春季, 与梨园梨火疫病发病规律相符。致病性测定结果表明, 气溶胶中分离的梨火疫病菌具有致病性。     

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.