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.     

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.     

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.     

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.     

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.     

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.     

梨火疫病的进境风险分析

讨论了梨火疫病的世界分布、国内苹果和梨的生产状况及经济重要性、梨火疫病在国内的适用性及国内外检测技术现状；澄清了以前我国有梨火疫病分布的错误报道。梨火疫病对我国梨和苹果生产具有极大的潜在风险，同时对我国的环境美化和生物多样性亦具潜在威胁。我国大部分地区为梨火疫病的适生区，国内广布梨火疫病的寄主。针对进口苗木的风险最大及果实亦可传病，提出了相应的风险管理措施。     

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.     

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.     

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,...     

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.     

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.     

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.     

利用蜜蜂传播生防菌防治梨火疫病

梨火疫病由梨火疫病菌（Erwinia amylovora）引起,可以通过蜜蜂授粉传播而加重危害。为做好香梨安全授粉工作,防止因直接放蜂造成梨火疫病的大流行,本研究测试了香梨花上分离的一株解淀粉芽孢杆菌（Bacillus amyloliquefaciens）对梨火疫病菌的拮抗效果,并利用自行研发的“蜜蜂导向盒”进行了蜜蜂传播生防菌防病试验。结果表明“蜜蜂导向盒+生防菌”技术在香梨花期可以有效控制由梨火疫菌引起的花腐和枝枯。     

利用Monte-Carlo模拟再评估梨火疫病病菌随水果果实的入侵风险

以梨火疫病随进境苹果果实传入的可能性为例，利用Monte-Carlo模拟方法评估有害生物的入侵风险。按照4种不同场景分别建立B分布来拟合入境水果中感染梨火疫病的比率，利用Pert分布拟合进口量。模拟表明，在4种场景下，最可能出现病害的时间分别为4702、3458、216和105年，在第4种场景即对入境苹果果实无任何检疫要求的情形下，出现发病的最少年份为14年。结果表明，传入风险随入境数量的增加而增加。     

Characterization of a Viral EPS-Depolymerase, a Potential Tool for Control of Fire Blight

ABSTRACT A 3.3-kb fragment of genomic DNA from bacteriophage Phi-Ea1h encoding an amylovoran-directed depolymerase lyase was sequenced, and three open reading frames (ORFs) were detected. The first ORF could encode a lysozyme and the second a holin that may form a pore supporting cell lysis by the lysozyme. The third ORF encodes a protein of 657 amino acids and deletion mutation in this DNA fragment abolished extracellular polysaccharide (EPS)-degrading activity. A putative promoter and a ribosome binding sequence were located in front of the gene. A polymerase chain reaction product spanning the gene was inserted into multi copy plasmids including fusions with a Histidine-tagged sequence to facilitate its purification on a nickel nitrilotriacetic acid column. Maximal activity of the purified protein was observed between pH 4 and 5 at 52 degrees C. Visualized by staining with fluorescein isothiocyanate-labeled lectin from Abrus precatorious, the enzyme degraded the EPS-capsules of Erwina amylovora. In virulence assays, no symptoms were detected for a low inoculum of an E. amylovora strain when pear slices were soaked in a solution of depolymerase in contrast to control slices without addition of the enzyme. Furthermore, gfp- or lux-labeled E. amylovora cells were not propagated, when their amylovoran capsules were removed by the depolymerase. The enzyme could be a tool for biological control of fire blight.     

Duplex real-time polymerase chain reaction reveals competition between Erwinia amylovora and E. pyrifoliae on pear blossoms

Erwinia amylovora and E. pyrifoliae are the causative agents of fire blight and Asian pear blight, respectively. The pathogens are closely related, with overlapping host ranges. Data are unavailable on the current distribution of E. pyrifoliae and on the interaction between the two species when they are present together on the same host. In this study, a duplex real-time polymerase chain reaction (PCR) protocol was developed to monitor the population dynamics of E. amylovora and E. pyrifoliae on the surface of Bartlett pear blossoms. Bacterial cells washed from blossoms were used directly as the PCR template without DNA extraction. Primers and a probe based on the E. amylovora levansucrase gene detected all E. amylovora strains. All E. pyrifoliae strains, including the Japanese Erwinia strains previously described as E. amylovora, were detected with a primer and probe combination based on the E. pyrifoliae hrpW gene. Disease development and severity were not significantly different in blossoms inoculated with individual Erwinia species or with a mixture of the two species. However, E. amylovora grew to greater population sizes than did E. pyrifoliae in both single species inoculations and in mixtures, suggesting that E. amylovora has a greater competitive fitness on Bartlett pear blossoms than E. pyrifoliae.     

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.