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.     

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.     

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.     

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.     

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.     

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.     

新疆库尔勒香梨火疫病的发生特点及防控建议

梨火疫病(fire blight)是由解淀粉欧文氏菌Erwinia amylovora引起的蔷薇科果树毁灭性病害。2017年首次在新疆巴音郭楞蒙古自治州发生,对库尔勒香梨产业造成严重危害和经济损失。本文总结5年来库尔勒香梨火疫病发生危害特点,详细描述了病害的田间症状表现,提出了防治建议,并对当前库尔勒香梨火疫病防治中存在的问题进行了讨论。     

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...     

Erwinia amylovora populations resistant to oxolinic acid in Israel: prevalence, persistence and fitness

Oxolinic acid (OA) has been the only bactericide used against fire blight in pear and quince orchards in Israel since 1998. OA-resistant Erwinia amylovora strains (Ea-OA^R) were detected in several orchards in two restricted areas in the northern Galilee region during 1999–2001. In the following years, resistant strains could not be detected in some of these locations. Documenting the fate of Ea-OA^R strains in commercial orchards at eight sites in northern Israel during 2000/03 revealed that the resistant population appeared irrespective of the number of sprays applied and the severity of the disease. The persistence of the Ea-OA^R populations varied from site to site, ranging from 4 to 20 months; these differences could be attributed to the fire blight management activities of growers. Comparative studies on the fitness of Ea-OA^R and E. amylovora strains sensitive to OA (Ea-OA^S) were conducted in vitro and in planta using two strains of each group. In four of the six comparisons, disease incidence on detached blossoms inoculated with Ea-OA^S was significantly higher than that on blossoms inoculated with Ea-OA^R. In two experiments conducted on 8-year-old pear trees grown under netting, the colonization of Ea-OA^S in blossoms, annual shoots and perennial spurs was significantly higher than that of the Ea-OA^R. In two experiments conducted on 2-year-old trees grown under netting in an experimental station, the incidence of shoots exhibiting fire blight symptoms and the rate of symptom progress within the branches were significantly higher in trees inoculated with Ea-OA^S than in those inoculated with Ea-OA^R. The results of this study suggest that OA-resistant E. amylovora strains have lower fitness than wild-type strains. These findings may have implications for fire blight management.     

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.     

Assessment of Environmental Factors Influencing Growth and Spread of Pantoea agglomerans on and Among Blossoms of Pear and Apple

ABSTRACT We evaluated effects of both physical and biological components of the environment on growth of Pantoea agglomerans on inoculated pear and apple blossoms and on spread of the bacterium to blossoms on non-inoculated trees. The center three rows of 0.35- to 0.5-ha blocks of four pear cultivars and four apple cultivars were sprayed with a suspension of streptomycin-resistant P. agglomerans strain C9-1S (C9-1S) at 20 to 60% and 60 to 90% bloom. Cultivars were chosen to create a sequence of continuous bloom from late March (d'Anjou pear) through mid-May (Red Rome apple). Each cultivar block was quartered into plots; two plots were treated twice with streptomycin sulfate near mid- and full bloom to suppress populations of indigenous bacterial epiphytes and the other two plots were treated with water. Colonization of blossoms by C9-1S and by indigenous bacterial epiphytes were monitored on inoculated trees and along transects of noninoculated trees. Immediately after spraying, C9-1S was detected principally on blossoms sampled from inoculated trees. As bloom progressed, trees up to 18 m from inoculated trees had high proportions of blossoms colonized by C9-1S. Streptomycin significantly (P 相似文献   

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.     

The interacting effects of temperature,duration of wetness and inoculum size on the infection of pear blossoms by<Emphasis Type="Italic">Erwinia amylovora</Emphasis>, the causal agent of fire blight

Pear blossoms are the plant tissue that is most vulnerable to infection byErwinia amylovora (Burrill) Winslowet al., the causal agent of fire blight. The interacting effects of temperature, wetness duration and inoculum size on the development of fire blight symptoms in detached pear blossoms were determined in three sets of experiments conducted under controlled conditions. It was expected that this information would facilitate the improvement of a warning system used in fire blight management. Results of the ANOVA tests of the data revealed highly significant interactions among the factors tested. The factors that contributed most to disease incidence were temperature and inoculum size; effects of wetness duration were significant in some cases, but that effect was small. It was further demonstrated that the effects of the interaction of these factors on the incidence of blossom infection may be understood in terms of the general concept of compensation. According to this concept, conditions highly favorable for one of the factors essential for pathogen development may compensate for other factors, for which the conditions are less favorable. As a result of the complex interactions observed between the biotic and abiotic factors, because of compensation relationships and because some of the factors cannot be estimated adequately (for example, inoculum level), it was concluded that it is not yet possible to improve fire blight management by using data on the quantitative relationships between biotic and abiotic factors. Contribution No. 532/02 from the Agricultural Research Organization. http://www.phytoparasitica.org posting Feb. 20, 2003.     

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。     

Analysis of Aggressiveness of Erwinia amylovora Using Disease-Dose and Time Relationships

ABSTRACT The aggressiveness of an extensive collection of strains of Erwinia amylovora was analyzed using immature fruit and detached pear flower assays under controlled environmental conditions. The analysis was performed by means of a quantitative approach based on fitting data to mathematical models that relate infection incidence to pathogen dose and time. Probit and hyperbolic saturation models were used for disease-dose relationships and provided information on the median effective dose (ED(50)). Values of ED(50) ranged from 10(3) to 10(6) CFU/ml (10 to 10(4) CFU per site of inoculation). A modified Gompertz model was used for disease-time relationships and provided information on the rate of infection incidence progression (r(g)) and time delayed to start of the incidence progress curve (t(0)). Values of r(g) ranged from near 0 to 1.90, and t(0) varied from 1.3 to more than 10 days. The more aggressive strains showed high r(g), low ED(50) values, and short t(0), whereas the less aggressive strains showed low r(g), high ED(50), and long t (0). The aggressiveness was dependent on plant material type and pear cultivars and was significantly different between strains of E. amylovora. Infectivity titration and kinetic analysis of progression of incidence of infections using the immature pear test and a standardized scale are proposed for assessment of strain aggressiveness. The implications of r(g), ED(50), and t(0) for the epidemiology and management of fire blight are discussed, particularly the wide range of aggressiveness among strains, the degree of host specificity observed in pear isolates, the very high infective potential of this pathogen, the independent action of pathogen cells during infection, and the possible advantage of including aggressiveness parameters into fire blight risk forecasting systems.     

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.     

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.     

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.     

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.     

Temporal Dynamics of the Biocontrol Agent Pseudomonas fluorescens Strain A506 in Flowers in Inoculated Pear Trees

ABSTRACT The colonization of individual flowers in mature pear orchards by Pseudomonas fluorescens strain A506 applied at different times during bloom was measured to determine the receptivity of flowers to colonization and the extent of intra-tree movement over time. Strain A506 populations in flowers open at inoculation were initially about 10(4) cells per flower and increased to approximately 10(6) cells per flower in flowers that were inoculated within about 5 days of opening. However, eventual populations decreased with further increases in flower age at inoculation to as few as about 10(3) cells per flower when inoculated flowers were more than 10 days old. Populations of strain A506 on flowers that opened after inoculation was initially very low at the time of petal expansion (<100 cells per flower) but increased rapidly with time after flower opening. The maximum population of strain A506 that developed on such flowers decreased with increasing time between inoculation and petal expansion; 10(4) to 10(5) cells of strain A506 eventually colonized flowers that opened within 7 days of inoculation, whereas fewer than 100 cells colonized flowers that opened 24 days or more after inoculation. Large total bacterial populations on A506-treated trees were associated with significant reductions in populations of Erwinia amylovora and reduced incidence of fire blight and severity of fruit russet.