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.     

Analysis of Variable Short-sequence DNA Repeats on the 29 kb Plasmid of Erwinia amylovora Strains

The fire blight pathogen Erwinia amylovora has been specifically and sensitively detected by PCR assays with primers derived from plasmid pEa29. The amplified fragment of approximately 1kb can vary in length for individual strains, easily seen in a digest with restriction enzymes Sau3A or HpaII. DNA fragments from this variable region were cloned and DNA sequence analysis revealed short-sequence DNA repeat (SSR) motifs which were reiterated to various extents. The SSR units consisted of eight nucleotides (ATTACAGA), and terminated with ATTA which is part of an SSR. The shortest repetition consisted of four units and the longest one in Austrian E. amylovora strains was 15 units. The number of SSR units was remarkably stable during propagation of strains, but was occasionally changed when a strain was stressed by exposure to antibiotics, copper sulphate or storage at low temperature. Changes in the SSR number could be due to adjustment in bacterial fitness to environmental pressure. We designed oligonucleotide PCR primers from DNA sequences adjacent to the SSR region of pEA29 for rapid analysis of SSR length variations. With this PCR assay, more than 130 strains were classified into at least 11 types based on the number of repeats. E. amylovora strains isolated in Germany carried mostly six repeats in pEA29, which never changed under laboratory conditions. E. amylovora strains from Hungary and the Netherlands were quite divergent for the SSRs and further changes were sometimes observed after plating on agar medium. Homology search of nucleotide sequence data libraries revealed similarities of the SSR motif to partition functions of low copy number plasmids. Amino acid homology searches showed similarity of the deduced amino acid sequence in the ORF adjacent to the SSR motif to replication proteins of plasmids. The SSR may play a role in regulation of plasmid replication and partition as assumed for iterons.     

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.     

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。     

Survival of Erwinia amylovora on pears and on fruit containers in cold storage and outdoors

The survival of Erwinia amylovora during cold storage or outdoors may be a relevant factor in the spread of fireblight. The survival of E. amylovora was studied in cold storage on pear fruits, on container materials and on packaging paper, and outdoors on wood (oak and poplar) and on polyethylene. The samples were contaminated with a bacterial suspension of a mutant strain, washed, concentrated by centrifugation, and the final concentrates were used for plate counting. In cold storage, reisolation from the calyx was successful even after 101 days, whereas on pear surfaces, it was unsuccessful after just 1 day. On oak and poplar wood, reisolation was obtained up to 77 days in cold storage for both types of wood, but only up to 27 and 55 days, respectively, outdoors. Reisolation from packaging paper in cold storage was successful up to 14 days. Reisolation from polyethylene outdoors was unsuccessful after 24 h. Survival curves were calculated for each material. On the basis of a model of inoculum transmission, and using the survival curves, a phytosanitary risk period for the different types of materials was estimated.     

Quantitative detection and monitoring of Colletotrichum acutatum in strawberry leaves using real-time PCR

Real-time PCR assays for Colletotrichum acutatum , one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) and the β-tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The β-tubulin-based assay was c . 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0·001% of the tissue was infected by C. acutatum . Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum . This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.     

Polymerase chain reaction fingerprinting of Erwinia amylovora has a limited phylogenetic value but allows the design of highly specific molecular markers

Erwinia amylovora, the causal agent of fire blight, is genetically very homogeneous, and current methodologies provide insufficient or contradictory information about the probable dispersal routes of the pathogen. With the final aim to obtain specific and reliable molecular markers for different lineages of the pathogen, we studied the molecular basis of rep-polymerase chain reaction (PCR) polymorphism using seven different arbitrary primers to fingerprint 93 E. amylovora strains from different countries, including Spain. Polymorphism was very low, and was displayed by only 11 E. amylovora strains, which produced 22 polymorphic bands. Five of 11 polymorphic bands cloned contained DNA that was present in more than 85% of the strains, whereas six bands were due to DNA present exclusively in the strains producing the rep-PCR polymorphism. Also, five of the polymorphic bands were due to the possession of either the ubiquitous plasmid pEA29, of plasmid pEU30, which was exclusively found in strains from North America, or of a 35-kb cryptic plasmid, present only in 28 strains from Northern Spain. We designed primer pairs from several cloned polymorphic bands that allowed the specific identification of the strains producing the polymorphism. Our results indicate that rep-PCR is not adequate for constructing genealogies of E. amylovora, although the strategy illustrated here, as well as the designed primers, can be used effectively in epidemiological studies with this pathogen.     

PCR-based detection of Colletotrichum acutatum on strawberry

An oligonucleotide primer ( Ca Int 2) was synthesized from the variable internal transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) from Colletotrichum acutatum . PCR with primers Ca Int2 and ITS4 (from a conserved sequence of the rDNA) amplified a 490 bp fragment from several isolates of C. acutatum but not from other members of the genus Colletotrichum . Amplification of this fragment was achieved from 100 fg of fungal DNA. These primers amplified a fragment of the same size from DNA extracted from strawberry tissues infected by C. acutatum . Southern hybridization analysis confirmed the 490 bp fragment from C. acutatum DNA and infected strawberry to be identical. The species-specific primer ( Ca Int2) developed in this work could be used for the accurate identification of C. acutatum and its detection on other host plants.     

Real-time Scorpion-PCR detection and quantification of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> on pear leaves and flowers

A specific primer couple (E3–E4) amplifying a single DNA fragment of 111 bp from plasmid pEA29 was designed to identify, detect and quantify Erwinia amylovora by real-time Scorpion-PCR. Specificity of primers and probe was assessed both by means of BLAST analyses and by using genomic DNA from a large number of E. amylovora isolates and other bacteria. In Scorpion-PCR, the limit of detection was of 1 pg of total DNA and a high correlation (r = 0.999) was achieved between target DNA quantity and cycle threshold (Ct). Combining two sequential amplifications with conventional reported primers (PEANT1–PEANT2) and Scorpion primers (E3 Scorpion-E4) the detection limit was of 1 fg (nested Scorpion-PCR). Using serial dilution of the bacterial suspensions the limit of detection was 3.2 × 10⁴ CFU ml⁻¹ in Scorpion-PCR and 2.8 × 10² CFU ml⁻¹ in nested Scorpion-PCR. Real-time PCR combined with effective procedures for DNA extraction enabled the detection and the quantification of the epiphytic population of E. amylovora in the washings of flowers and leaves of artificially inoculated pear. A significant correlation (r = 0.92) was achieved between pathogen CFU on semi-selective media and the corresponding target DNA concentration evaluated by real-time PCR.     

Molecular characterization of Erwinia amylovora strains from different host plants through RFLP analysis and sequencing of hrpN and dspA/E genes

A total of 73 Erwinia amylovora strains obtained from 13 Maloideae host species and from Rubus spp., and isolated from different geographic areas, were assessed using RFLP and DNA sequencing analysis of the 3' hrp N gene and/or of a fragment of 1341 bp of the dsp A/E region. An Erwinia pyrifoliae strain, used as outgroup, was checked in the same way. For the three strains isolated from Rubus spp. and for one strain from Amelanchier sp., RFLP analysis of the hrp N gene using the Rsa I enzyme yielded a PCR product 60 bp smaller than that of all the other strains. Sequence analysis of the gene revealed this was due to the absence of a 60 bp fragment in the noncoding region downstream of the gene. The strain PD 2915, isolated from Amelanchier sp. grown in Canada, showed five same-sense substitutions and one missense substitution at position 868 of the hrp N gene, converting aspartic acid into asparagine. Also, restriction analysis of a fragment of 613 bp of the dsp A/E region with Cfo I revealed an RFLP pattern suitable for differentiating the E. amylovora strains isolated from Rubus spp. and Amelanchier sp. from all the others. In the dsp A/E coding region, the four strains showed 13–14 missense point mutations, in some cases yielding drastic amino acid substitutions. In addition, partial sequencing of the dsp A/E region of PD 2915 from Amelanchier sp. indicated a higher similarity to E. amylovora strains isolated from Rubus spp. than towards strains from other Maloideae hosts. The E. pyrifoliae strain showed 23 single nucleotide substitutions along the hrp N gene and 88% of nucleotide identity with E. amylovora strains in the portion of dsp A/E region. Artificial inoculations on immature pear fruits and young shoots of Maloideae and Ruboideae showed a restricted pathogenicity for the strains from Rubus and Amelanchier , with the latter inciting blight symptoms only on Amelanchier .     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.     

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.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

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.     

Erwinia pyrifoliae, an Erwinia species different from Erwinia amylovora, causes a necrotic disease of Asian pear trees

Bacteria from necrotic branches of Asian pear trees ( Pyrus pyrifolia ) in Korea were consistently isolated as white colonies on nutrient agar and formed mucoid, slightly yellow colonies on a minimal medium with copper sulphate. Isolates with this colony morphology were studied in a series of microbiological, molecular and pathological tests. Most isolates allowed the verification of Koch's postulate on P. pyrifolia seedlings and on slices from immature pear ( Pyrus communis ) fruits and were also positive in hypersensitivity tests on tobacco leaves. They showed characteristics common to species in the genus Erwinia , but were different from Erwinia amylovora , the agent of fire blight. A relationship between the novel pathogen and E. amylovora was found in microbiological and serological tests. Both organisms had similar but not identical protein patterns in 2-D gel electrophoresis, and in growth morphology the new pathogen produced colonies on MM2 Cu medium that were mucoid and slightly yellow, compared with the clearly yellow colonies of E. amylovora . No similarity was found in the plasmid profiles, and consequently no PCR signal was obtained with primers from the E. amylovora plasmid pEA29. REP-PCR also produced bands differing for the two organisms.     

Conventional PCR and real time quantitative PCR detection of <Emphasis Type="Italic">Phytophthora cryptogea</Emphasis> on <Emphasis Type="Italic">Gerbera jamesonii</Emphasis>

A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1 gene of P. cryptogea. A 369 bp product was amplified on DNA from 17 isolates of P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared. The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5 × 10³ P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the re-circulating nutrient solution of closed soilless cultivation systems.     

玉米圆斑病病原的快速检测

玉米生平脐蠕孢菌(Bipolaris zeicola)是引起玉米圆斑病的病原菌。本研究通过对玉米生平脐蠕孢菌及其近似种的EF-1α基因(elongation factor 1α)部分序列进行比对,设计出玉米生平脐蠕孢菌的特异性引物Y-EF-F和Y-EF-R,利用该引物可以从B.zeicola中扩增出137 bp的特异片段,而其余的17个参试菌株扩增结果为阴性。灵敏度实验表明该对引物可以检测到目标DNA的浓度为1 pg·μL~(-1)。用B.zeicola接种玉米叶片、苞叶以及玉米粒,然后以接种发病的病组织DNA为模板,利用引物Y-EF-F和Y-EF-R进行PCR扩增,可以扩增出137 bp的特异性条带,而健康玉米组织DNA中未能扩增出任何条带。用B.zeicola孢子悬浮液接种大田玉米叶片,接种第3 d可以检测到未发病组织中有B.zeicola病原菌,第5 d可以看到明显的病斑。研究结果表明该方法可用于快速、准确和灵敏地检测玉米组织中的潜伏期玉米生平脐蠕孢菌,为玉米圆斑病的快速检测,进而及早采取防治措施提供积极的指导。     

Use of the DNA sequence of the intergenic spacer region between the 16S and 23S rRNA genes for the identification of Clavibacter michiganensis subsp. insidiosus at the molecular level

A study has been performed to identify Clavibacter michiganensis subsp. insidiosus at the molecular level, using the polymerase chain reaction (PCR) technique with oligonucleotide primers based on specific sequence recognition of the intergenic spacer region between the 16S and 23S rRNA genes. The pair of primers was designed on the basis of available DNA sequence data for that region in C. m. insidiosus and other bacteria. Using this pair of primers, a large amount of an amplified DNA fragment of 218 bp in length was obtained from C. m. insidiosus. The specificity of this amplification was proved by PCR analysis, using the above-mentioned pair of primers and templates from different bacteria, some related to C. m. insidiosus. The PCR products were analysed using agarose gel electrophoresis.     

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.     

异源Harpin对软腐欧氏杆菌与植物互作关系的影响

 本文通过三亲交配将带有梨火疫欧文氏杆菌功能完整的hrp基因簇的重组粘粒pCPP430转移到了胡萝卜软腐欧氏杆菌(Erwinia carotovora) Se9R中。从接合子中提纯的质粒与pCPP430的大小、酶切图谱相同;以该质粒为模板,用针对hrpN序列的引物经PCR扩增得到与hrpN大小相同的片段;用地高辛标记的hrpN作为探针进行Southern blotting分析,在接合子中的质粒上得到预期的杂交带。接合子可以在烟草叶片上稳定地引起过敏反应。另外,接合子可以在寄主马铃薯叶片上引起过敏反应样枯斑,表明异源harpin的表达及功能不受软腐菌的抑制。该接合子在体外产生的果胶酸裂解酶活性与受体菌Se9R无明显差别。低浓度的接合子在马铃薯块茎上的软腐症状明显低于受体菌Se9R。外源harpin表达可能不改变软腐欧氏杆菌的致病力、而是通过诱导马铃薯抗性达到减轻发病的作用。