Antibiosis and acidification by Pantoea agglomerans strain E325 may contribute to suppression of Erwinia amylovora

Pantoea agglomerans strain E325, a commercially available antagonist for fire blight of apple and pear, was originally selected through screening based on suppression of Erwinia amylovora on flower stigmas, but specific mechanisms of antagonism were unknown. Bacterial modification of pH was evaluated as a possible mechanism by analyzing stigma exudates extracted from 'Gala' apple stigmas. The pH values for field samples were only slightly lower than controls, but indicated a range (pH 5 to 6) conducive for antibiotic activity according to subsequent assays. Under low-phosphate and low-pH conditions, an antibacterial product of E325 with high specificity to E. amylovora was effective at low concentrations. A minimum of 20 to 40 ng of a ninhydrin-reactive compound purified using RP-HPLC caused visible inhibition in assays. Activity was heat stable and unaffected by amino acids, iron, or enzymes known to affect antibiotics of P. agglomerans. Antibiosis was diminished, however, under basic conditions, and with increasing phosphate concentrations at pH 6 and 7. Inhibition was not observed in media containing phosphate concentrations commonly used in antibiosis assays. We propose that E325 suppresses the fire blight pathogen not only by competing for nutrients on the stigma, but by producing an antibiotic specific to E. amylovora. Further work is necessary to substantiate that the compound is produced and active on flower stigmas.     

Temperature and Pomaceous Flower Age Related to Colonization by Erwinia amylovora and Antagonists

ABSTRACT Fire blight of apple and pear is initiated by epiphytic populations of Erwinia amylovora on flower stigmas. Predicting this disease and managing it with microbial antagonists depends on an understanding of bacterial colonization on stigmas. Detached 'Manchurian' crab apple flowers were inoculated with E. amylovora and subjected to a range of constant temperatures or various fluctuating temperature regimes. Results may have application to disease risk assessment systems such as the Cougarblight model, which now are based on in vitro growth of the pathogen. In other experiments, detached crab apple flowers and attached 'Gala' apple flowers were maintained at different temperatures for various periods before inoculation with E. amylovora or antagonists (Pseudomonas fluorescens strain A506 and Pantoea agglomerans strains C9-1 and E325). Maximum stigma age supporting bacterial multiplication decreased as temperature increased, and was reduced by pollination. Stigmas were receptive to bacteria at ages older than previously reported, probably due to less interference from indigenous organisms. The study revealed antagonist limitations that possibly affect field performance (e.g., the inability of strain A506 to grow on relatively old stigmas conducive to the pathogen). Such deficiencies could be overcome by selecting other antagonists or using antagonist mixtures in the orchard.     

Effect of nectar on microbial antagonists evaluated for use in control of fire blight of pome fruits

ABSTRACT Under warm, dry conditions, Erwinia amylovora can become established in relatively high populations on apple (Malus domestica) or pear (Pyrus communis) flower stigmas, and subsequent wet conditions facilitate its movement to the flower hypanthium where infection generally is initiated through the nectarthodes. Research on biological control of fire blight has focused mainly on the flower stigma, and knowledge is lacking regarding the effect of nectar on microbial antagonists in the flower hypanthium. The biocontrol agents Pseudomonas fluorescens strain A506 and Pantoea agglomerans strain C9-1 were cultured in a basal liquid medium with various concentrations (0 to 50% total sugar) of sucrose or synthetic nectar (sucrose/glucose/fructose, 2:1:1). Strain A506 showed less growth and lower survival than strain C9-1 at high sugar levels, and A506 was less effective than C9-1 as a preemptive antagonist of E. amylovora in high-sugar media. Both antagonist strains were less tolerant to high sugar levels than E. amylovora (strain Ea153). The same bacteria were cultured in a medium with 25% total sugar consisting of various proportions of sucrose, glucose, and fructose, and growth response correlated strongly with solute potential. When 28 microbial strains were cultured in synthetic nectar (25% total sugar) and ranked based on growth, strains clustered according to taxonomic group. Yeasts were most osmotolerant, followed by strains of E. amylovora, Pantoea agglomerans, Bacillus spp., and Pseudomonas spp. Further studies done in planta are necessary to determine whether osmotolerance of antagonists is advantageous in the biological control of fire blight.     

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.     

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.     

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.     

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.     

Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees

Bacteria from necrotic branches of Asian pear trees ( Pyrus pyrifolia ) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear ( Pyrus communis ) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia , but were different from Erwinia amylovora , the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2-D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora . No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP-PCR also produced bands differing for the two organisms.     

An Indigenous Virulent Strain of Erwinia amylovora Lacking the Ubiquitous Plasmid pEA29

ABSTRACT An atypical strain of Erwinia amylovora was isolated near an outbreak of fire blight at a nursery in Spain in 1996. It was obtained from a Crataegus plant showing typical symptoms and was identified as E. amy-lovora by biochemical tests and enrichment-enzyme-linked immuno-sorbent assay, but not by polymerase chain reaction using primers based on the pEA29 sequence. Nevertheless, with primers from chromosomal regions, the isolate gave the expected amplification band. This strain carries one plasmid of approximately 70 kb, with no homology with the 29-kb plasmid common to all pathogenic strains, or with a large plasmid present in some E. amylovora strains. Growth of the strain in minimal medium without thiamine was slower compared with cultures in the same medium with thiamine, a characteristic typical of strains cured of the 29-kb plasmid. Nevertheless, aggressiveness assays on pear, apple, and Pyracantha plants and in immature pear fruit showed that this strain exhibited a virulence level similar to other strains containing pEA29. To the best of our knowledge, this is the first report of the isolation from naturally infected plant material of a pathogenic strain of E. amylovora without pEA29, but with a plasmid of approximately 70 kb not previously described.     

An Extracellular Protease of Pseudomonas fluorescens Inactivates Antibiotics of Pantoea agglomerans

ABSTRACT Pseudomonas fluorescens A506 and Pantoea agglomerans strains Eh252 and C9-1 are biological control agents that suppress fire blight, an important disease of pear and apple caused by the bacterium Erwinia amylovora. Pseudomonas fluorescens strain A506 suppresses disease largely through competitive exclusion of E. amylovora on surfaces of blossoms, the primary infection court, whereas Pantoea agglomerans strains Eh252 and C9-1 produce antibiotics that are toxic to E. amylovora. In this study, an extracellular protease produced by A506 is characterized and evaluated for its capacity to inactivate the antibiotics produced by the strains of Pantoea agglomerans. Activity of the extracellular protease was optimal at pH 9 and inhibited by zinc- or calcium-chelators, indicating that the protease is an alkaline metalloprotease. In an agar plate bioassay, partially purified extracellular protease inactivated the antibiotics mccEh252 and herbicolin O, which are produced by Pantoea agglomerans strains Eh252 and C9-1, respectively. Derivatives of A506 deficient in extracellular protease production were obtained by transposon mutagenesis, and the aprX gene encoding the protease was cloned and sequenced. Strain A506 inactivated mccEh252 and herbicolin O in agar plate bioassays, whereas the aprX mutant did not inactivate the antibiotics. Both A506 and the aprX mutant were insensitive to antibiosis by C9-1 and Eh252; thus, the protease was not required to protect A506 from antibiosis. These data highlight a previously unknown role of the extracellular protease produced by Pseudomonas fluorescens A506 in interactions among plant-associated microbes.     

Antibiosis Contributes to Biological Control of Fire Blight by Pantoea agglomerans Strain Eh252 in Orchards

ABSTRACT Fire blight, caused by Erwinia amylovora, is the most serious bacterial disease of pear and apple trees. Biological control with strains of Pantoea agglomerans (syn. Erwinia herbicola) may provide an effective disease management strategy for fire blight. Most strains of P. agglomerans evaluated for suppression of fire blight produce compounds that inhibit the growth of E. amylovora in culture. The role of these inhibitory compounds in fire blight suppression in orchard environments has not been studied. In seven field trials in Oregon, we compared the population dynamics and disease suppression with P. agglomerans Eh252, a strain that produces a single antibiotic, with its near-isogenic antibiotic-deficient derivative, strain 10:12. Water or suspensions of Eh252 or 10:12 (1 x 10(8) CFU/ml) were applied at 30 and 70% bloom to pear or apple trees. Aqueous suspensions of freeze-dried cells of E. amylovora (3 x 10(5) CFU/ml) were applied at full bloom. Additional trees were treated with streptomycin or oxytetracycline at 30 and 70% bloom and in some experiments, 1 day after application of the pathogen. Population sizes of Eh252 or 10:12 on pear blossoms were estimated by spreading dilutions of blossom washes on culture media. Average population sizes of Eh252 and 10:12 on blossoms ranged from 10(5) to 10(7) CFU, and in five of six trials, the relative area under the population curve of Eh252 was not significantly different than that of its derivative 10:12. Both Eh252 and 10:12 reduced the growth of the pathogen on blossoms compared with inoculated water-treated controls. Eh252 significantly decreased the incidence of fire blight in six of seven field trials compared with the incidence on water-treated trees, and 10:12 similarly reduced the incidence of fire blight in four of seven trials. In three of seven field trials, trees treated with Eh252 had a significantly lower incidence of fire blight compared with trees treated 3 with 10:12. Overall,3 Eh252 reduced the incidence of fire blight by 55 +/- 8%, 10:12 by 30 +/- 6%, streptomycin by 75 +/- 4%, and oxytetracycline by 16 +/- 14%. The effectiveness of strain 10:12 compared with water treatment indicates that other mechanisms (e.g., competitive exclusion or habitat modification) also contribute to disease suppression by P. agglomerans. The increased suppression of fire blight by the parental strain Eh252 compared with the antibiotic-deficient mutant 10:12 indicates that antibiosis is an important mechanism of biological control of fire blight.     

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.     

Bioavailability of Iron to Pseudomonas fluorescens Strain A506 on Flowers of Pear and Apple

ABSTRACT The addition of 0.1 mM FeCl(3) to a defined culture medium induces the bacterial epiphyte Pseudomonas fluorescens strain A506 (A506) to produce an antibiotic toxic to the fire blight pathogen, Erwinia amylovora. Consequently, because A506 is registered and applied as a commercial product to suppress E. amylovora before floral infection of pear and apple, the relative availability of iron to A506 on surfaces of pear and apple flowers is of potential significance. An 'iron biosensor' construct of A506 was developed by transformation with an iron-regulated promoter (pvd) fused to a promoterless ice nucleation reporter gene (inaZ). This construct, A506 (pvd-inaZ), established high populations on pear and apple flowers, ranging from 10(4) to 10(6) CFU/flower. In seven trials on pear and apple trees, A506 (pvd-inaZ) expressed high ice nucleation activity (INA) on flowers, indicating limited iron bioavailability or a low-iron environment unlikely to induce antibiotic production by A506. A506 (pvd-inaZ) also colonized flowers when mixed with chemicals containing iron: FeSO(4) or the iron chelates ferric ethylenediaminedi-(o-hydroxyphenyl-acetic) acid (FeEDDHA) and ferric diethylenetriamine pentaacetate (FeDTPA). These compounds represent an array of commercial iron formulations applied to foliage to avert iron chlorosis. Treatment of flowers with a mixture of A506 (pvd-inaZ) and 3 mM FeEDDHA or FeDTPA significantly decreased INA compared with flowers treated with A506 (pvd-inaZ) in water. Lower concentrations (0.3 mM) of FeEDDHA, however, did not consistently suppress INA. These results indicate that apple and pear flowers represent an iron-limited environment to A506 and that treatment with 3 mM FeEDDHA is needed to increase significantly the level of iron available to this bacterium.     

Eop1 from a Rubus strain of Erwinia amylovora functions as a host-range limiting factor

Strains of Erwinia amylovora, the bacterium causing the disease fire blight of rosaceous plants, are separated into two groups based on host range: Spiraeoideae and Rubus strains. Spiraeoideae strains have wide host ranges, infecting plants in many rosaceous genera, including apple and pear. In the field, Rubus strains infect the genus Rubus exclusively, which includes raspberry and blackberry. Based on comparisons of limited sequence data from a Rubus and a Spiraeoideae strain, the gene eop1 was identified as unusually divergent, and it was selected as a possible host specificity factor. To test this, eop1 genes from a Rubus strain and a Spiraeoideae strain were cloned and mutated. Expression of the Rubus-strain eop1 reduced the virulence of E. amylovora in immature pear fruit and in apple shoots. Sequencing the orfA-eop1 regions of several strains of E. amylovora confirmed that forms of eop1 are conserved among strains with similar host ranges. This work provides evidence that eop1 from a Rubus-specific strain can function as a determinant of host specificity in E. amylovora.     

Blue EaLAMP—a specific and sensitive method for visual detection of genomic Erwinia amylovora DNA

Fire blight caused by the gram-negative bacterium Erwinia amylovora is a major disease of pome fruit like apple (Malus domestica) or pear (Pyrus communis). Detection of the pathogen is hampered by low titres usually being present during initial flower infection and the brief time frame for analysis to decide upon subsequent countermeasures. Here we describe a loop-mediated isothermal amplification of DNA (LAMP) assay for genomic DNA of Erwinia amylovora, which relies on a highly specific primer design that excludes amplification from typical DNA sources of the orchard biological system and from sample handling. The assay enables the fast detection of down to approximately 20?cfu of pure Erwinia amylovora or 100?fg genomic DNA (corresponding to approximately 25 E. amyolovora equivalents) per reaction within 45?min. Fast and reliable detection of E. amylovora in orchard samples by naked eye is achieved through a visual colour change indication with hydroxynaphthol blue. The assay avoids use of complex technical devices and is thus suitable for field testing.     

The Role of Water in Epiphytic Colonization and Infection of Pomaceous Flowers by Erwinia amylovora

ABSTRACT Detached crab apple flowers were used as an experimental model to investigate the effect of relative humidity (RH), free moisture, and water potential Psi(w) on the interaction between Erwinia amylovora and pomaceous flowers. Flowers were maintained at 24 degrees C with the cut pedicel submerged in a sucrose solution. The bacterium multiplied on inoculated flower stigmas at between approximately 55 and 100% RH but not in the floral cup (hypanthium) until the RH was higher than 80%. To study the effect of free moisture, stigma-inoculated flowers were kept wet for different periods. Flowers became diseased only with wetting, and incidence was high (77%) even when water application was immediately followed by a 52-min drying period. In other experiments with hypanthium-inoculated flowers, RH or sucrose concentration in holding vials was varied to affect Psi(w) of flower nectar and ovary tissue. Population size of E. amylovora in the hypanthium increased with nectar Psi(w) following a sigmoidal curve (R(2) = 0.99). Disease incidence and severity, however, were more closely related to ovary Psi(w) (R(2) = 0.85 and 0.91, respectively) than to bacterial population size (R(2) = 0.25 and 0.67, respectively) as fitted to the quadratic equation. Maximum disease incidence and severity occurred at an ovary Psi(w) above -2.0 MPa, and disease severity continued to increase above -1.0 MPa. These results were confirmed with detached flowers of Delicious apple and d'Anjou pear. A practical implication is that disease might be partly managed in arid climates by limiting soil irrigation water during bloom and early fruit set.     

Establishment of Bacterial Antagonists of Erwinia amylovora on Pear and Apple Blossoms as Influenced by Inoculum Preparation

The influence of inoculum preparation on the establishment of bacterial antagonists that suppress fire blight and Erwinia amylovora on blossoms was evaluated. Aqueous suspensions of Pseudomonas fluorescens A506, E. herbicola C9-1R, or E. amylovora 153N were prepared from cells harvested from the surface of an agar medium or from cells that were lyophilized after culture under similar conditions. Bacterial suspensions (1 x 10(8) CFU/ml) were sprayed on pear and apple trees at 50% bloom near midday. The incidence of recovery (proportion of blossoms containing detectable populations) and the population sizes of the bacteria on individual blossoms with detectable populations were followed over a period of several days. Fluorescent microspheres (1 mum in diameter) were added to sprays at a concentration of 1 x 10(7) microspheres per ml to mark blossoms that were open during application of bacteria. After dilution-plating, the stigmas and styles of each blossom were examined for the presence of microspheres with an epifluorescence microscope. In three of five trials, bacteria applied as suspensions of lyophilized cells were recovered from a greater proportion of blossoms than bacterial cells harvested directly from culture media. Every blossom harvested within 6 days after spraying had microspheres present on the surfaces of the styles and stigmas; thus, lack of establishment of detectable populations, rather than escape of blossoms from spray inoculation, accounted for the differences in proportion of blossoms colonized by the different preparations of bacteria. The use of lyophilized cells in field trials decreased variability in the establishment of bacteria on blossoms.     

Testing a Rapid Diagnostic Medium for Erwinia amylovora and Development of a Procedure for Sampling Blossoms in Pear Orchards

ABSTRACT The coliform agar produced by Merck was tested for rapid diagnosis of Erwinia amylovora (the causal agent of fire blight) in pear blossoms. The medium enabled the diagnosis to be completed within 36 h. Diagnoses performed with the medium were confirmed by the BIOLOG and the fatty-acid profile methods. The diagnostic medium was used to determine the spatial distribution of colonized blossoms in the orchards and it was found that E. amylovora may be distributed both in clusters and at random. These findings were used in the development of a statistical model for sampling blossoms in the orchard. The model determines the number of trees to be sampled in the orchard and the number of blossoms be taken from each tree, which would enable the true colonization incidence of blossoms in the orchard to be estimated at desired levels of accuracy and confidence. Parameters included in the model are: the total number of trees in the orchard (T), the number of trees to be sampled in the orchard (t), the number of blossoms to be sampled from each tree (n), the true colonization incidence of blossoms (pi), a coefficient of aggregation (rho), the required level of confidence (1 - alpha), and the required level of accuracy (L). Sensitivity analyses revealed that the parameter governing sample size is the required level of accuracy. Sampling of 20 blossoms from each of several hundred trees is required to achieve an accuracy of +/-1%, but only a few single trees are needed for an accuracy level of +/-10%. A sampling procedure then was developed, validated with an independent data set, and found to be accurate. It was concluded that sampling of pear blossoms and estimation of the incidence of blossom colonization by E. amylovora could improve fire blight management, but not in all cases.     

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。     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。