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.     

Yeasts as antagonists against fireblight

Yeasts are potential antagonists of microorganisms in the phyllosphere. Due to their osmotolerance, they should also be able to colonize apple flowers. In field experiments, we applied yeast agents against fireblight at two different sites in the southern part of Germany. They showed efficiencies 0–20% below Plantomycin (streptomycin). Co-culture experiments in liquid basal media with synthetic nectar resulted in suppression of Erwinia amylovora by yeast. This effect could not be confirmed with population studies of yeasts and E. amylovora in flower clusters. Field and laboratory experiments indicated that yeasts have antagonistic properties against fireblight but further research is needed to investigate this potential.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Histology of waxflower (Chamelaucium spp.) flower infection by Botrytis cinerea

Botrytis cinerea infects waxflower (Chamelaucium spp.) flowers and can induce them to abscise from their petioles before disease becomes evident. Botrytis cinerea infection of flowers was studied on two waxflower cultivars by light and electron microscopy. Pot‐grown waxflower flowers were harvested, inoculated with aqueous suspensions of B. cinerea conidia, incubated at 20–22°C and >95% RH and examined within 96 h post‐inoculation (hpi). Conidial germination on petals started 4 hpi, penetration via germ tube tips was 6 hpi and protoappressoria were formed 8 hpi. Germination on petals approximately doubled every 4–6 h to 18 hpi. Conidial germination was ca. 50% at 22–24 hpi. Botrytis cinerea infected most waxflower flower organs, including petals, anthers and filaments, stigma and hypanthium, within 24 hpi. Hyaline and lobate appressoria were observed 36 hpi. Infection cushions on stamen bases were formed 36 hpi by saprophytic hyphae that originated from anthers. This infection process can give rise to tan‐coloured symptoms typical of botrytis disease that radiate from this part of the flower. Subcuticular hyphae were present at high density near stamen bases and evidently resulted from multiple penetrations from single infection cushions. The subcuticular hyphae grew within the hypanthium and towards the centre of the floral tube. When flower abscission occurred, floral tube tissues close to the abscission zone remained uninfected. This observation infers possible transmission of a signal (e.g. ethylene) upon B. cinerea infection. Thus, B. cinerea causes flower abscission apparently as a defence response.     

The influence of inoculum concentration, relative humidity, and temperature on infection of greenhouse tomatoes by Botrytis cinerea

Botrytis cinerea causes serious crop losses in greenhouse tomato crops through infection of flowers and stem wounds. Experiments were carried out to determine the effects of inoculum concentration, relative humidity (RH), and temperature at these two infection sites. Infection of permanent flower parts increased as a function of inoculum concentration and both length of exposure to high RH (approximately 100% for 0–36 h) and specified continuous RH (56–100%). A low level of infection was still evident under continuous 56% RH. Interruption of periods of high RH with breaks of low RH did not reduce infection. Infection of stem wounds was less dependent on inoculum concentration or RH. Factorial combinations of inoculum concentration, RH, and temperature produced significant interactions. Higher temperature increased infection of flowers but reduced infection of stem wounds. The main implications for control in commercial crops are as follows. Lowering the aerial spore concentration by maintaining the disease at a low level will reduce flower infection. Lowering RH will reduce but not eliminate flower infection but will have only a small effect on stem infection. Raising the temperature (from 15 to 25°C) will reduce stem infection, and whilst flower infection increases, this is counteracted by increased flower production and a decrease in the proportion of infections reaching the peduncle and stem.     

Relationships Between Blueberry Flower Age, Pollination, and Conidial Infection by Monilinia vaccinii-corymbosi

ABSTRACT Monilinia vaccinii-corymbosi infects open blueberry flowers via the gynoecial pathway, leading to mummification of the developing fruit. To determine the effect of flower age on infection, stigmata were inoculated with conidia of M. vaccinii-corymbosi between 0 and 5 days after anthesis, fungal growth rates through the stylar canal were measured in detached flowers in the laboratory, and fruit disease incidence was determined in plants grown in the greenhouse. Hyphal growth rates were greatest in flowers inoculated on the day of anthesis, declined linearly with increasing flower age at inoculation (r = 0.921; P < 0.0001; n = 12), and were unaffected by the presence or absence of pollen applied at the time of inoculation. In greenhouse-grown plants, the percentage of infected fruit decreased exponentially with increasing flower age at inoculation (R = 0.878; P = 0.0057; n = 10), with disease incidence ranging from 76.4% for flowers inoculated on the day of anthesis to 15.5% for those inoculated 4 days later. Fruit disease incidence in the greenhouse was linearly correlated with hyphal growth rates in detached flowers (r = 0.985; P < 0.0001; n = 9), justifying the use of detached flowers when investigating gynoecial infection by M. vaccinii-corymbosi. In separate experiments, the effects of timing and sequence of pollination and inoculation on hyphal growth rates through the stylar canal and on disease incidence were investigated. Application of pollen to detached flowers 1 or 2 days before inoculation reduced hyphal growth rates by between 14.0 and 42.9% compared with flowers that received pollen and conidia simultaneously. Similarly, reductions in fruit disease incidence by between 9.5 and 18.3% were observed on greenhouse-grown plants for pollination-to-inoculation intervals ranging from 1 to 4 days. These results document that newly opened flowers are most susceptible to infection by M. vaccinii-corymbosi and that fruit disease incidence is reduced if pollination occurs at least 1 day before inoculation. Strategies that lead to early pollination of newly opened flowers may be useful for managing mummy berry disease in the field.     

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.     

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.     

龙爪槐花对异色瓢虫成虫寿命的影响

本文研究了龙爪槐花和其所含的主要糖类对异色瓢虫成虫寿命的影响。室内用龙爪槐花饲养的异色瓢虫雌、雄成虫平均寿命分别为10.96和10.83 d,显著长于龙爪槐叶上成虫寿命(雌:3.98 d,雄:3.65 d);室外龙爪槐花序上生长的异色瓢虫成虫平均寿命(雌性:14.72 d,雄性:15.46 d)也显著高于枝条上的个体(雌:3.90 d,雄:3.82 d)。补充葡萄糖、果糖、蔗糖和麦芽糖4种糖液,能显著延长异色瓢虫成虫寿命。龙爪槐花含糖量较高的花粉与花蜜是异色瓢虫成虫的适宜食物、能维持其种群发生,是花期龙爪槐上虽没有蚜虫发生、但仍有高密度异色瓢虫成虫的主要原因。     

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.     

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

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

Influence of incubation-period humidity on the development of brown rot blossom blight of sour cherry

ABSTRACT When detached sour cherry (Prunus cerasus) blossoms were inoculated with conidia of Monilinia fructicola and subjected to a standard 8-h wetting treatment at 20 degrees C, blossom blight incidence was proportional to relative humidity (RH) when RH was held constant during the subsequent 6-day incubation period (frequency = 1.0 at the maximum RH of 92%; frequency = 0.38 at the minimum RH of 57%). Similarly, when a primary incubation period at 87% RH was followed by a secondary incubation period at 54% RH, blossom blight incidence was proportional to the number of hours at the higher level (frequencies of 0.94, 0.80, and 0.38 with primary incubation periods of 6 days, 36 h, and 12 h, respectively). When intact blossoms on potted trees were exposed to common inoculation and wetting treatments, disease incidence was consistently high on trees that subsequently were incubated in a controlled environment chamber (20 degrees C, 90 to 95% RH) but was extremely variable when trees were incubated under variable ambient conditions. Ambient incubation temperature had little effect on disease incidence 9 days after inoculation, whereas ambient RH had a pronounced effect: the frequency of blighted blossoms was 0.53 to 0.61 when the number of hours at RH >90% was approximately two to six times that at RH <60%, whereas this frequency was only 0.02 to 0.07 when the number of hours at RH >90% was approximately one-third the number at RH <60%. After 48 h at a constant RH of 89 or 57%, the water potential of excised uninoculated blossoms was -1.15 and -1.93 MPa, respectively; however, growth of M. fructicola on osmotically adjusted potato dextrose agar was unaffected by changes in water potential within this range. Thus, although RH during incubation has an important influence on blossom blight development, the causal mechanism remains uncertain.     

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.     

Heat treatment for the control of rose and carnation grey mould (Botrytis cinerea)

Thermal dip treatment for flower heads was found effective against grey mould of roses. Dipping of flowers in tap-water at 50 C for 20 40 s was more effective than at higher or lower temperatures and than for longer treatments. The treatment was effective in five out of six rose cultivars and in one carnation cultivar. A 60, reduction in disease severity following the treatment was observed in flowers naturally infected by Botrytis cinerea, whereas the treatment was not effective against artificial infection of flowers. Conditioning of flowers for 2 days at 10 C and high relative humidity before incubation under grey-mould-conducive conditions (15 C. high humidity) increased the efficacy of the thermal treatment. Moreover, the combination of heat with 3 mM catechol was additively more efficient in reducing grey mould. The surfactant Tween 20 (001 %) improved the effect of treatment at 45 C for 20 s. A combination of heat with the fungicide chlorothalonil did not improve effectiveness, whereas when polyoxin B was combined with heat it was better than either treatment alone. The temperature of petals at the edge and centre of flower reached 37 and 27 C, respectively, at the end of a 20-s incubation period in 50 C water. Conidia treated at 34-40 C for 10-20 s incited 60% less severe disease than conidia treated at 25 C in water.