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The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease., (© 2013 BSPP AND JOHN WILEY & SONS LTD.)

Microencapsulation and controlled release of the biocontrol agent Pantoea agglomerans strain E325 (E325), an antagonist to the bacterial plant pathogen Erwinia amylovora that causes fire blight, a devastating disease of apple and pear, have been investigated. Uniform core-shell alginate microcapsules (AMCs), 60-300 μm in diameter, were fabricated to encapsulate E325 within the core, along with nutrients, to preserve viability and promote proliferation. Controlled release of E325 was achieved by separately adjusting alginate concentrations in the shell and core solutions, and by modifying the AMC size. Viability of E325 was monitored via fluorescent staining, revealing either lack of or minimal stress during or after encapsulation. Proliferation of E325 within AMCs, followed by their subsequent release, and colonization activities within confines of apple flowers were studied under different encapsulation conditions using rfp-labeled E325 to obtain highly promising results. This study provided a 'proof of concept' of the successful use of a microencapsulated biocontrol agent, E325, against E. amylovora, and could serve as a model for further studies on the development of effective plant disease management strategies., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.

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.

RcsC is a hybrid sensor kinase which contains a sensor domain, a histidine kinase domain, and a receiver domain. We have previously demonstrated that, although the Erwinia amylovora rcsC mutant produces more amylovoran than the wild-type (WT) strain in vitro, the mutant remains nonpathogenic on both immature pear fruit and apple plants. In this study, we have comparatively characterized the Erwinia RcsC and its homologs from various enterobacteria. Results demonstrate that expression of the Erwinia rcsC gene suppresses amylovoran production in various amylovoran overproducing WT and mutant strains, thus suggesting the presence of a net phosphatase activity of Erwinia RcsC. Findings have also demonstrated that rcsC homologs from other enterobacteria could not rescue amylovoran production of the Erwinia rcsC mutant in vitro. However, virulence of the Erwinia rcsC mutant is partially restored by rcsC homologs from Pantoea stewartii, Yersinia pestis, and Salmonella enterica but not from Escherichia coli on apple shoots. Domain-swapping experiments have indicated that replacement of the E. coli RcsC sensor domain by those of Erwinia and Yersinia spp. partially restores virulence of the Erwinia rcsC mutant, whereas chimeric constructs containing the sensor domain of E. coli RcsC could not rescue virulence of the Erwinia rcsC mutant on apple. Interestingly, only chimeric constructs containing the histidine kinase and receiver domains of Erwinia RcsC are fully capable of rescuing amylovoran production. These results suggest that the sensor domain of RcsC may be important in regulating bacterial virulence, whereas the activity of the histidine kinase and receiver domains of Erwinia RcsC may be essential for amylovoran production in vitro.

Apple blossoms were sampled for indigenous epiphytic populations of culturable microorganisms during different stages of bloom at two locations in central Washington State and one site in Corvallis, OR. Frequencies and population sizes of bacteria on stigmas of apple were lower in Washington than at Corvallis, where average relative humidity was higher and possibly favored greater colonization; however, bacteria at Corvallis were mainly pseudomonads, whereas those in Washington were diverse, composed of several genera. In Washington, yeast as well as bacteria were isolated from both stigmatic and hypanthial surfaces. Sampled blossoms were processed immediately to assess microbial populations, or after a 24-h incubation at 28 degrees C and high relative humidity, which broadened the range of detectable taxa evaluated as potential antagonists. Identifications were based on fatty acid methyl ester profiles and rDNA sequence analyses. Yeasts or yeastlike organisms were detected at frequencies similar to or greater than bacteria, particularly in hypanthia. When microbial isolates were tested for their capacity to suppress Erwinia amylovora on stigmas of detached crab apple flowers, many were ineffective. The best antagonists were the bacteria Pantoea agglomerans and Pseudomonas spp. and a few yeasts identified as Cryptococcus spp. Further evaluation of these taxa on flowers could lead to the discovery of additional biocontrol agents for fire blight.

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.

Blossom age as related to hypanthial susceptibility to Erwinia amylovora is not well established, but is relevant to disease risk assessment. To test this, detached crab apple blossoms were maintained for various periods and at different temperatures before applying inoculum to hypanthia. Inoculum potential on hypanthia due to wetting was evaluated by subjecting detached stigma-inoculated blossoms (~10 6 CFU per flower) to varying amounts and durations of simulated rain (or dew) at 14°C. Blossoms of varying age on mature 'Gala' apple trees were inoculated on hypanthia with 10 2 , 10 4 , or 10 6 CFU per flower. In the laboratory, susceptibility decreased with flower age at rates that increased with temperature. Wetness periods up to 12 h resulted in populations on hypanthia of <10 3 CFU per flower; 24 h of wetness resulted in ~10 4 or ~10 5 CFU. A dose response was shown in the orchard, and regression curves indicated steepest decline of susceptibility during initial days after petal expansion. Disease models incorporating a blossom-age component may be effective because they indicate the potential for infection when temperatures favor rapid bacterial growth on stigmas within a window of high hypanthial susceptibility. Further investigation of these relationships could lead to advancements in determining fire blight risk.

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.

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.

The efficacy of Pantoea agglomerans strain E325 for control of fire blight of apple was determined in comparative field trials involving other bacterial antagonists. Concurrently, the importance of the natural dispersal of bacteria as a complicating factor was assessed. Tests were performed under two sets of conditions, those that allowed for the dispersal of bacteria via honeybee activity and those that minimized it through the use of translucent polyethylene enclosures around single trees. The enclosures also raised daytime temperatures and allowed for controlled wetting, two factors important to the development of blossom blight. Singleantagonist treatments with strain E325, Pseudomonas fluorescens strain A506, and P. agglomerans strain C9-1 were applied to open blossoms on each of 10 enclosed trees and 10 nonenclosed trees. During bloom, suspensions of antagonists (10 8 CFU/ml) were applied twice with a brush, and a suspension of Erwinia amylovora (10 7 CFU/ml) was subsequently applied once using the same method. Two days after inoculation with the pathogen, trees were misted to simulate precipitation. Flower-to-flower spread of antagonistic bacteria was less frequent on trees surrounded by plastic enclosures than on nonenclosed trees. The range and statistical separation of means for population size of E. amylovora and disease incidence among treatments were greater for enclosed trees than for nonenclosed trees. Based on these results, the natural spread of antagonists being compared may mask differences in their efficacy as biocontrol agents. Such distortions and resulting misinterpretations could be lessened by separating treatments widely in large orchard blocks and by monitoring microbial populations. Strain E325 from fresh or lyophilized cultures was consistently more effective than standard antagonists in suppressing E. amylovora and reducing disease incidence. To fully assess its potential use for fire blight, larger-scale trials under various conditions will be necessary.

ABSTRACT The relationship of cumulative chill-hours (hours with a mean temperature <7.2 degrees C) and heating degree-days (base 7.2 degrees C) to carpogenic germination of pseudosclerotia of Monilinia vaccinii-corymbosi, which causes mummy berry disease of blueberry, was investigated. In two laboratory experiments, pseudosclerotia collected from rabbiteye blueberry in Georgia were conditioned at 5 to 6 degrees C for 26 to 1,378 h prior to placement in conditions favorable for germination and apothecium development. The number of chill-hours accumulated during the conditioning period affected the subsequent proportion of pseudosclerotia that germinated and produced apothecia, with the greatest incidence of carpogenic germination occurring after intermediate levels of chilling ( approximately 700 chill-hours). The minimum chilling requirement for germination and apothecium production was considerably lower than that reported previously for pseudo-sclerotia from highbush blueberry in northern production regions. The rate of carpogenic germination was strongly affected by interactions between the accumulation of chill-hours and degree-days during the conditioning and germination periods; pseudosclerotia exposed to prolonged chilling periods, once transferred to suitable conditions, germinated and produced apothecia more rapidly (after fewer degree-days had accumulated) than those exposed to shorter chilling periods. Thus, pseudosclerotia of M. vaccinii-corymbosi are adapted to germinate carpogenically following cold winters (high chill-hours, low degree-days) as well as warm winters (low chill-hours, high degree-days). Results were validated in a combined field-laboratory experiment in which pseudosclerotia that had received various levels of natural chilling were allowed to germinate in controlled conditions in the laboratory, and in two field experiments in which pseudosclerotia were exposed to natural chilling and germination conditions. A simple model describing the timing of apothecium emergence in relation to cumulative chill-hours and degree-days was developed based on the experiments. The model should be useful for better timing of field scouting programs for apothecia to aid in management of primary infection by M. vaccinii-corymbosi.

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.

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.

The application of ultraviolet light (UV-C, 254 nm) hormesis on fruits and vegetables to stimulate beneficial responses is a new method for controlling storage rots and extending the shelf-life of fruits and vegetables. The present study was aimed at treating tomatoes ( Lycopersicon esculentum ) with different UV-C dosages (1.3 to 40 KJ/m 2 ) to induce resistance to black mold ( Alternaria alternata ), gray mold ( Botrytis cinerea ), and Rhizopus soft rot ( Rhizopus stolonifer ). These diseases were effectively reduced when tomatoes were inoculated following UV-C irradiation. UV-C treated tomatoes were firmer in texture and less red in color than the control tomatoes, indicating a delay in ripening. Slower ripening and resistance to storage rots of tomatoes are probably related. The positive effect of UV-C on tomatoes decreased as treatments were performed at stages of increased ripeness.

Criconemella xenoplax, Fusarium solani, and F. oxysporum caused necrosis of Nemaguard peach feeder roots in greenhouse tests. Root necrosis was more extensive in the presence of either fungus than wtih C. xenoplax alone. Shoot growth and plant height were less for plants inoculated with F. oxysporum or F. solani than for plants inoculated with the fungi plus C. xenoplax. Neither synergistic nor additive effects on root necrosis or plant growth occurred between C. xenoplax and the fungal pathogens.