梨火疫病在欧美国家的发生规律及防治措施

梨火疫病是严重危害梨和苹果的流行性重要和世界性植物检疫对象,至今该病尚未传入中国。本文对梨火疫病在欧美国家的发生历史,症状识别,危害特征,流行规律及防治措施等进行了重点介绍。     

梨火疫病菌噬菌体的初步研究

为找到梨火疫病菌适度专化性的噬菌体，用于该病的检测和鉴定，从英国病山楂枝条上分离到一株噬菌体，测定了其寄主范围、潜育期、繁殖量等，还观察了其形态。分离到的噬菌体为高度专化性噬菌体，不能单位用于梨火疫病菌的检测和鉴定，可与其他梨火疫病菌噬菌体株系配合使用。     

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。     

免疫吸附PCR技术提高梨火疫病菌检测灵敏度

本研究采用已经发表的引物和制备的梨火疫病菌抗血清,研制了免疫吸附-PCR技术,使其检测梨火疫病菌纯菌的灵敏度比标准PCR技术提高10倍;检测模拟样品中的梨火疫病菌灵敏度提高了1000倍;从相关混合菌液中能够更加灵敏和准确地检测出梨火疫病菌.该方法简单易行,准确灵敏,具有广阔的应用前景.     

免疫捕获PCR检测进境苹果果实中梨火疫病菌

利用梨火疫病菌抗体和PCR技术建立了梨火疫病菌免疫捕获PCR方法,检测苹果模拟样品的灵敏度达到了1.5×102cfu/reaction。与直接PCR进行比较,免疫捕获PCR方法检测苹果模拟样品的灵敏度提高了10倍以上,而且省去了DNA提取等步骤。利用该方法成功从进境苹果样品中检测到梨火疫病菌,产物测序结果表明,产物序列和梨火疫病菌的相应序列高度一致。试验结果表明,免疫捕获PCR法能除去样品中的大部分PCR反应抑制物质,可以有效检测进境苹果中梨火疫病菌,在口岸水果检疫中具有一定的应用潜力和推广价值。     

应用地理信息系统对梨火疫病可能分布区的初步研究

本文利用地理信息系统分析了梨火疫病在我国梨、苹果种植区的可能分布区和非分布区。根据梨、苹果的种植区域、梨火疫病的世界分布现状及梨火疫病的生物学特性，应用地理信息系统，分析确定了７月平均最高温度＜２３℃为其下限指标，５月份（北半球）或１１月（南）平均最高温度＞１４℃为上限指标，３－５月平均降水＞２ｍｍ为其经济分布的限制因子，初步确立梨火疫病在我国及世界的可能分布区。预测结果表明与该病实际分布相吻合。     

梨火疫病菌的实时荧光PCR检测

 根据梨火疫病菌16S~23S间的ITS保守序列,设计并合成了一对特异性引物REA/FEA,应用荧光染料SYBR Green I,对10个梨火疫的菌株和其它相关参试菌株进行了检测。结果表明,10个梨火疫菌株都产生荧光信号而其它参试菌株都不产生荧光信号,成功建立了梨火疫病菌的实时荧光PCR检测方法。整个检测过程只需3h,完全闭管,降低了污染的机会,无需PCR后处理。检测的灵敏度是4个菌体细胞,比常规PCR电泳检测提高了10倍。用该特异性引物对梨枝条浸泡液进行实时荧光PCR检测,结果可特异性检测到目标菌的存在,并且检测的灵敏度是24个菌体细胞,比常规PCR电泳检测提高10倍。     

介绍一种简易准确的梨火疫病检测方法

梨火疫病是梨和苹果树上一种危险的细菌性病害。它在美国、新西兰危害十分严重。1957年梨火疫病传入欧洲,近年已蔓及西欧沿海各国,我国至今末发现梨火疫病,梨火疫病是重要对外植物检疫对象之一,寻找一种准确、快速,简便易行的检测方法是我国当前对外检疫工作巾一个迫切需要解决的问题。对梨火疫病菌的选择性培养基研究自 Lelliott(1968)报道营养蔗糖选择性培养基以来相继已推出数种。它们都具有一定的应用价值,但最终证实还要依靠致病性测定。     

梨火疫病在地中海地区扩展

在北美、欧洲及世界其它一些地区,近几年来,梨火疫病(Erwia amglovora)在地中海地区已成为生产上的一大问题。据埃及植病学家报道,1982年和1983年该病造成严重损失,1984年又一次大发生。同年,塞浦路斯报道发生该病。1985年5—6月,巴勒斯坦地区(以色列)东部的约旦何谷地多处发现梨火疫病。据分析,该病来自塞浦路斯方向的自然盛     

进境苹果果实中梨火疫病菌的套式PCR检测

 针对进境商用苹果果实携带梨火疫病菌Erwinia amylovora数量有限的特点,选取源于病菌pEA29质粒的2对引物P29A/P29B和PEANT1/PEANT2配对组合成套式PCR,其检测灵敏度可达0.15 pg菌体DNA,检测灵敏度高于EPPO推荐的单管套式PCR方法和常规PCR方法。分别利用这3种PCR检测方法对美国、新西兰、日本和智利等国进境的166批苹果样品进行检测,3种检测方法的样品阳性率分别为53.6%、38.0%和8.4%,试验结果表明此套式PCR检测方法可用于进境商用苹果的梨火疫病菌快速检测。进境样品的检测结果证实了进境商用苹果果实中存在梨火疫病菌的可能性。     

Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction

Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross-recognized by the production of a weaker PCR band of similar size to E. amylovora . In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 10² cfu mL^–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora -spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora , the PCR detection sensitivity was determined to be 6.6 × 10² cfu mL^–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

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.     

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.     

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.     

梨园气溶胶中梨火疫病菌的定量检测

为系统研究梨园气溶胶中梨火疫病菌的含量, 本研究于2019年－2021年在新疆库尔勒市人和农场梨园, 利用病原菌孢子捕捉器在每年春季(4月下旬)、夏季(6月中旬)、秋季(9月中旬)收集梨园气溶胶, 检测梨火疫病菌。结果显示, 健康梨园气溶胶中未检测到梨火疫病菌, 不同发病程度的梨园气溶胶中均能检测到梨火疫病菌, 携菌量均值在102 cfu/(24cm2·h)以上, 其中, 气溶胶中梨火疫病菌含量最高值为2.81×104 cfu/(24cm2·h), 最低值为8.50×102 cfu/(24cm2·h); 重度、中度、轻度发病果园收集的气溶胶中含梨火疫病菌总菌落数均值分别为8.74×103、4.55×103、2.36×103 cfu/(24cm2·h)。此外, 在同一高度收集的气溶胶中, 梨火疫病菌菌落数随收集时间的延长而增加。不同季节气溶胶携菌量检测结果表明, 秋季发病梨园中气溶胶携菌量明显高于夏季和春季, 与梨园梨火疫病发病规律相符。致病性测定结果表明, 气溶胶中分离的梨火疫病菌具有致病性。     

Dual Activity of a Viral Lysozyme with High Efficiency for Growth Inhibition of Erwinia amylovora

ABSTRACT The lysozyme from Erwinia amylovora phage PhiEa1h was investigated for its ability to inhibit growth of bacteria and compared with the lysozyme from Escherichia coli phage T4. The assays to measure lysozyme activity included cell lysis and growth inhibition of bacteria. Bacterial strains with kanamycin resistance were not affected by lysates containing the PhiEa1h-enzyme. The titer of Micrococcus luteus but not of Erwinia amylovora was diminished by cell extracts containing T4 lysozyme. In contrast, PhiEa1h lysozyme preferentially inhibited E. amylovora, exceeding the T4 lysozyme activity at least one million-fold. Spherical cells were formed after application to E. amylovora similar to lyz-gene expression in Escherichia coli. Heating of cell extracts destroyed the murami-dase activity, but retained an antibacterial activity. Other plant-associated bacteria related to Erwinia amylovora also were inhibited for growth when cell extracts with PhiEa1h lysozyme were applied to soak pear slices and potato slices. Ooze formation and soft rot caused by E. amylovora or E. carotovora subsp. atroseptica, respectively, were strongly reduced and the PhiEa1h lysozyme was more efficient compared with extracts containing T4 lysozyme.     

Genetic Analysis of a Pathogenic Erwinia sp. Isolated from Pear in Japan

ABSTRACT Four Erwinia strains, originally isolated in Japan from pear trees with bacterial shoot blight symptoms, were analyzed to determine their genetic relationship with Erwinia amylovora and E. pyrifoliae. When genomes were characterized with amplified fragment length polymorphism markers and by comparative groEL sequence analysis, the Japanese Erwinia sp. and South Korean E. pyrifoliae strains were placed in the same group, which was phylogenetically distinct from a group of 15 strains of E. amylovora. Sequencing of the 29,593-bp plasmid pEJ30 from Erwinia strain Ejp556 revealed that this plasmid was nearly identical to plasmid pEP36 from E. pyrifoliae and was closely related to the nontransferable ubiquitous plasmid pEA29 from E. amylovora. Twenty-one presumptive genes and their order in pEP36 were highly conserved in pEJ30; however, transposon Tn5394, which was present in pEP36, was not found in pEJ30. Short-sequence DNA repeats were conserved between pEJ30 and pEP36, and were different from short-sequence repeats in pEA29. Despite base-pair mismatches, primer pairs used in pEA29 polymerase chain reaction assays for E. amylovora amplified plasmid DNA from the Japanese Erwinia Ejp556 and Ejp562. Like E. pyrifoliae and a few strains of E. amylovora, Japanese Erwinia Ejp617 contained plasmids related to E. pyrifoliae ColE1-related plasmid pEP2.6. Based on these genetic analyses, we conclude that the Erwinia pathogen of pear in Japan is closely related to E. pyrifoliae and that both of these pathogens are demonstrably distinct from E. amylovora.