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

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

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.     

Characterization of theErwinia amylovora population in Israel

A collection of 205 strains ofErwinia amylovora isolated in Israel over a period of 12 years has been established. The strains were isolated from different varieties of pear, apple, loquat and quince grown in Israel, and collected from different locations in the country. They were characterized in respect to degree of virulence on several hosts and serological and molecular characters. Pathogenicity tests carried out on flowering branches of pear and apple, shoots of pears, and on trees of pear and loquat grown in containers outdoors, revealed no significant differences in the severity of blossom blight or shoot blight among the various strains. ELISA and immunofluorescence assays revealed no serotypic groups among the Israeli strains. Genomic diversity was studied by random amplified polymorphic DNA (RAPD) analysis using 24 arbitrary 10-base primers. All the strains examined (45 Israeli and 11 from Egypt, Cyprus and Greece) produced the same RAPD patterns with each of the primers used. Amplification patterns were indistinguishable from those produced by strains isolated from the neighboring countries. Results presented in this study suggest that the population ofE. amylovora in Israel is homogenous.     

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.     

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,...     

Warning Systems for Fireblight1)

The ideal warning system should indicate conditions favouring inoculum production and dissemination, infection and subsequent symptom expression. It should give guidance on the best times for inspections to be made and for chemical control measures to be applied. The choice of system is governed by the crop, the climate and economic considerations. New systems must be developed to deal with new situations. Most previous systems have been concerned with the spring blossom periods of pear and apple, but systems for assessing fireblight activity at other times in the growing season are being developed. This is especially important in Europe where the blossom periods of susceptible ornamental hosts extend into July and where, in some countries, summer blossom on pear may be present throughout the season. Monitoring of inoculum levels on cankers, blossoms or shoots has proved valuable where economy in the use of sprays has justified its use; sprays are usually withheld until the pathogen is detected. Warning systems based on weather data are broadly in agreement but differ in detail. Sprays during bloom are often withheld until maximum temperatures exceed 18° C (or mean temperatures reach 14.5 —- 16.5° C) and there is precipitation or high humidity (> 60 % RH) at the same time. Some workers would spray immediately after moderate rain (e.g. 2.5 – 5.0 mm) when inoculum was known to be present. Spray applications immediately after damaging storms are the general rule. Proper evaluation of warning systems is difficult and is only possible in areas where fireblight is endemic and where adequate field records can be obtained.     

Risk Analysis of Brown Rot Blossom Blight of Prune Caused by Monilinia fructicola

Experiments under controlled environmental conditions were conducted during bloom of prune (Prunus domestica, L.) in 1999 and 2000 to assess the effects of inoculum concentration (IC), wetness duration (WD), temperature, and bloom stages on development of brown rot blossom blight of prunes. Branches from trees of a prune orchard were inoculated with Monilinia fructicola at different bloom stages and incubated at different temperatures with different periods of WD. The proportion of blighted blossoms (PBB) for each inoculated branch was determined. Bloom stage, IC, temperature, and WD significantly affected blossom blight of prunes. PBB at popcorn and full bloom stages was significantly greater than PBB at later bloom stages (P 30 degrees C and less than 4-h WD. However, longer than 4-h WD linearly increased incidence of blossom blight. A risk assessment table of blossom blight was produced for different environmental conditions to guide the control of prune brown rot.     

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。     

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   

A Statistical Comparison of the Blossom Blight Forecasts of MARYBLYT and Cougarblight with Receiver Operating Characteristic Curve Analysis

ABSTRACT Blossom blight forecasting is an important aspect of fire blight, caused by Erwinia amylovora, management for both apple and pear. A comparison of the forecast accuracy of two common fire blight forecasters, MARYBLYT and Cougarblight, was performed with receiver operating characteristic (ROC) curve analysis and 243 data sets. The rain threshold of Cougarblight was analyzed as a separate model termed Cougarblight and rain. Data were used as a whole and then grouped into geographic regions and cultivar susceptibilities. Frequency distributions of cases and controls, orchards or regions (depending on the data set), with and without observed disease, respectively, in all data sets overlapped. MARYBLYT, Cougarblight, and Cougarblight and rain all predicted blossom blight infection better than chance (P = 0.05). It was found that the blossom blight forecasters performed equivalently in the geographic regions of the east and west coasts of North America and moderately susceptible cultivars based on the 95% confidence intervals and pairwise contrasts of the area under the ROC curve. Significant differences (P < 0.05) between the forecasts of Cougarblight and MARYBLYT were found with pairwise contrasts in the England and very susceptible cultivar data sets. Youden's index was used to determine the optimal cutpoint of both forecasters. The greatest sensitivity and specificity for MARYBLYT coincided with the use of the highest risk threshold for predictions of infection; with Cougarblight, there was no clear single risk threshold across all data sets.     

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.     

运城市主要果树花期冻害指标研究

运城市主要果树近年来均有不同程度花期冻害发生,为减少冻害损失,制定更具生产指导意义的农业气象灾害指标以及农业气象灾害标准,文中以运城市主要种植果树苹果、梨、桃、杏花器官为研究对象,利用智能人工气候箱模拟自然降温进行研究.结果表明:运城市苹果花期、梨花期、杏花期遭受冻害临界温度均为-2℃,桃花期遭受冻害临界温度为-3℃;...     

性信息素迷向丝对不同果树梨小食心虫的防控效果

2017年在宁夏果园开展了性信息素迷向丝(北京中捷四方生物科技公司生产的长效迷向丝,有效成分占30%)对为害不同果树的梨小食心虫的防控效果研究。试验地共5.5 hm~2,栽植有成龄苹果树、梨树和李树。试验区按树种设置3个处理区,3个对照区。处理区在边缘区域每株树平均悬挂3条迷向丝,内部区域隔树悬挂,每树悬挂1条迷向丝,悬挂高度为距离地面2 m。调查各树种全年诱蛾量、诱蛾下降率、蛀果率等指标,研究迷向丝对不同果树上梨小食心虫的防控效果,分析苹果、梨和李树分区域集中栽培下梨小食心虫为害特点。研究结果显示:3个处理区的诱蛾下降率均达96%以上,果实膨大期蛀果下降率均达50%以上,成熟期蛀果下降率均达57%以上,说明性信息素迷向丝对3种果树区域的梨小食心虫防控效果明显且稳定;不同果树区域梨小食心虫发生高峰期一致;梨园中诱捕的梨小食心虫成虫最多,说明其具有寄主选择性;李园蛀果率明显高于苹果园和梨园,李果实成熟采摘后成虫诱捕量减少,同时期苹果园与梨园成虫诱捕量增加,说明梨小食心虫具有迁移为害特性。     

黄色黏虫板在3种果园对蚜虫及其天敌的诱集作用

为减少黄色黏虫板(简称黄板)对果园害虫天敌的影响,明确黄板在果园中的使用方法,于2014年5月4日至9月30日在山西省太谷县的苹果、梨、桃园中进行了黄板诱集蚜虫及其天敌的动态调查。结果表明:在3种果园黄板可以诱集8目32科的昆虫,主要类群为蚜虫、叶蝉、瓢虫、食蚜蝇、草蛉和蚜茧蜂等。在苹果园和桃园中诱蚜量呈双峰谷线状,在梨园中呈三峰状;各果园寄生性天敌的数量变化趋势与蚜虫一致且有跟随效应,但苹果园和梨园诱集的捕食性天敌均在8月下旬至8月底出现高峰期,而桃园中捕食性天敌水平一直较高。3个果园在花期至幼果期即5月4日至6月22日益害比最小,苹果园仅为1:202.8,而在其它时段的益害比均大于1:150,甚至最高可达1:19.4,表明在果园中使用黄板的害处大于益处,建议在果园中慎用黄板诱蚜。     

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。     

Effectiveness of preventing flowering of hawthorn in protecting pear orchards from fire blight infection

Since 1984 when a new Ministerial Regulation on fire blight came into force, there have been 20 protected regions in the Netherlands, where nurseries of rosaceous plants, and pear and apple orchards are extra protected against fire blight. This policy is also necessary to meet the requirements of the European Community (EC) on fire blight. Two of the measures in the protected regions are the prohibition of flowering of the native hawthorn (Crataegus monogyna andC. laevigata), and destruction of blighted plants. In the unprotected regions, flowering is allowed, and destruction of blighted plants is limited to a zone of 500 metres around orchards.For three years, the effectiveness of preventing flowering of hawthorn in protecting pear orchards against fire blight infection was studied in the field. Five test areas of about 3 km × 3 km were chosen with hawthorns and pear orchards. Two of these areas were in protected regions and three in unprotected regions. The more than 50 000 hawthorns in the areas were grouped into 1125 sites of hawthorn. The 126 orchards larger than 0.2 ha contained about 180 000 trees.During the three years light to moderate epidemics of fire blight were observed in the regions. Fire blight occurred in 2.3% of the non-flowering sites and 19.8% of the flowering (or fruiting) sites at least once in 1987, 1988 or 1989. The prohibition of flowering for hawthorn in protected areas was rather well implemented, so that in protected areas a smaller proportion of sites of hawthorns had fire blight (4.1%) than in unprotected areas (14%). Moreover, there were fewer sites per square kilometre in the protected areas (13) than in the unprotected areas (26).In protected areas, 53% and in unprotected areas 59% of the pear orchards had fire blight during 1987, 1988 or 1989. The difference was not significant. The first reason for the ineffectiveness of the preventing of flowering prevention in hawthorn to control fire blight in pear orchards was the inadequate hygiene of the pear orchards in both types of region. If it be assumed that a new focus is most probably initiated by the nearest existing focus, the second reason was that fire blight hardly spread from hawthorn to pear in the period of this study. Spread of fire blight within pear orchards and between pear orchards occurred frequently.     

Levels of fire blight (<Emphasis Type="Italic">Erwinia amylovora</Emphasis>) susceptibility of native apple,pear and quince germplasm from Lake Van Basin,Turkey

Fire blight resistance of apple, pear and quince genetic resources from Lake Van Basin (eastern Turkey) was tested using Erwinia amylovora strain Ea Van. Shoot tips of 92 native accessions (48 accessions for apple, 38 accessions for pear and 6 accessions for quince) were wounded for inoculation, and artificially inoculated with pathogenic bacteria under greenhouse conditions. The levels of resistance of accessions were classified in comparison with control varieties according to the genotype susceptibility index (GSI%) scores based on the lesion length on shoots of each genotype. Fire blight resistance of accessions consisted of five classes: resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). GSI% scores differed significantly among accessions from each fruit species (p < 0.01). GSI values ranged from 12.4% to 64.1% for apple genotypes, from 17.2% to 55.1% for pear genotypes, and from 17.8% to 43.4% for quince genotypes. No resistant genotypes of apple, pear and quince were observed. Seven accessions of apple, two accessions of pear and one accession of quince were MR. 25 accessions of apple, 14 accessions of pear and one accession of quince were MS. These findings indicate a considerable variation in fire blight resistance and could contribute to breeding efforts regarding fire blight resistance in apple, pear and quince.     

Bait twig method for soil detection of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>, causal agent of white root rot of Japanese pear and apple,at an early stage of tree infection

Mulberry twigs were inserted into the soil as bait to detect Rosellinia necatrix at an early stage of tree infection in the orchard. R. necatrix was frequently trapped on twigs near the trunk base at soil depths of 6–20 cm within 10–20 days in May–July, suggesting that the incubation period was dependent on soil temperature. Subsequently, we inserted twig in the soil around healthy-looking trees in naturally infested orchards. R. necatrix was trapped from 80.0% of Japanese pear and 75.0% of apple trees that later proved to be infected. This bait twig method facilitated quicker diagnosis of white root rot on Japanese pear and apple at early stages of infection and can be used to detect recurrence of the fungus after fungicide treatment.     

Biological control of fire blight of hawthorn (Crataegus monogyna) with Erwinia herbicola under protected conditions

Isolates of Erwinia herbicola , obtained from flowers and leaves of hawthorn ( Crataegus monogyna) , were screened as potential control agents of fire blight disease (caused by Erwinia amylovora) using an immature pear fruit assay. Selected isolates were subsequently tested for disease control by infection of hawthorn blossom in the laboratory, and by shoot infection of hawthorn plants grown under controlled (glasshouse) and fluctuating (polythene tunnel) environmental conditions.
Although the immature pear fruit assay provided a general screen for the selection of antagonists for the control of both blossom and shoot blight, it had two major limitations when quantitatively applied. Firstly there were inconsistencies in the relative effects of different isolates on the pear-slice surface, with some isolates being more suppressive than the standard antagonist Eh252 in the first screening and less in the second. Secondly the assay was not able to predict accurately the level of control in the intact plant-as no correlation occurred between the level of control in the pear fruit assay and the percentage control of either blossom blight or shoot blight.
Two isolates of E. herbicola , WL9 and WL40, reduced both blossom- and shoot-blight. WL9 provided over 80% control of blossom blight, equivalent to that provided by chemical agents, and also gave total control of shoot blight when applied at a WL9: pathogen ratio of 10:1.