Erwinia isolates from the bacterial shoot blight of pear in Japan are closely related to Erwinia pyrifoliae based on phylogenetic analyses of gyrB and rpoD genes

The phylogenetic relationships among Erwinia amylovora biovar 4 (the pathogen of bacterial shoot blight of pear in Japan), other biovars of E. amylovora, and Erwinia pyrifoliae were investigated using the sequences of 16S rRNA, gyrB, and rpoD genes. The tested isolates formed two distinct monophyletic groups in the phylogenetic trees constructed based on the gyrB gene, rpoD gene, or a combination of the three genes: group 1 contained E. amylovora biovars 1, 2, and 3; group 2 contained E. amylovora bv. 4 and E. pyrifoliae. This phylogenetic analysis showed that E. amylovora bv. 4 was more closely related to E. pyrifoliae than to other biovars of E. amylovora. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB242876 to AB242925.     

Phenotypic and genetic characterization of Erwinia rhapontici isolated from diseased Asian pear fruit trees

In 1995, a serious necrotic disease appeared in Asian pear trees in the orchards of Chuncheon, South Korea. Large numbers of bacterial samples were collected, and the causative microorganism was identified as a novel pathogen, Erwinia pyrifoliae. Among the samples, four bacterial isolates with atypical characteristics were further analyzed through biochemical tests and 16S rRNA gene sequence analysis. By phenotypic and genetic analysis these isolates were identified as E. rhapontici. Phylogenetic analysis using 16S rRNA sequence data revealed that E. rhapontici forms a discrete clade with high bootstrap value close to the E. amylovora group. Pathogenicity tests on leaves of tobacco plants (Nicotiana tabacum) elicited hypersensitivity responses, but artificial inoculations on immature fruits and shoots of Asian pear (Pyrus pyrifolia) did not show any necrotic symptoms. The developed primers showed no cross reactivity when tested against other phytopathogens and were able to detect E. rhapontici from mixed culture and in planta.     

Taxonomic Position of the Causal Pathogen of Bacterial Shoot Blight of Pear

Bacteriological properties and DNA-DNA homology values were compared among the pathogen causing bacterial shoot blight of pear (BSBP) isolated in 1994–1996, Erwinia amylovora isolated outside of Japan, and other Amylovora group bacteria. Bacteriological properties of BSBP strains were identical to those of E. amylovora in the majority of tests, but differed distinctively in several tests, including hydrolysis of esculin and acid production from salicin, etc. BSBP strains differed from the others in the Amylovora group in many other tests. DNA homology among the strains of BSBP ranged from 85 to 103% and from 83 to 110% among strains of E. amylovora. In contrast, the values between BSBP strains and E. amylovora strains were 55 to 81%, while those between BSBP strains and other Amylovora group strains were 42% or less. We consider, therefore, that the BSBP pathogen may well be included in E. amylovora at the species level. E. amylovora, including BSBP strains, however, can be classified into four biovars based on differences in nine tests such as growth factor requirements and crater formation on high sucrose medium. Namely, there are two biovars from Maloideae sources, one from Rubus idaeus, and one from the source of BSBP in Hokkaido. The presence of these biovars suggests a correlation with geographical, serological, and pathogenic differentiations in the species of E. amylovora. The BSBP pathogen in Hokkaido was identified as E. amylovora bv. 4 which is distinct from E. amylovora bv. 1, 2 and 3 isolated in countries outside of Japan. Received 29 July 1999/ Accepted in revised form 12 October 1999     

Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan

Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.     

Complete sequence of Erwinia piriflorinigrans plasmids pEPIR37 and pEPIR5 and role of pEPIR37 in pathogen virulence

Erwinia piriflorinigrans is a newly described pathogen causing necrosis of pear blossoms. Complete sequencing of the 37‐kb plasmid pEPIR37 common to 27 E. piriflorinigrans strains revealed homology to sequences of the ubiquitous plasmids pEA29 of the fire blight pathogen E. amylovora, plasmid pEP36 of E. pyrifoliae, plasmid pEJ30 of Erwinia sp. from Japan, and genomic regions of the related Rosaceae epiphytic Erwinia species E. tasmaniensis and E. billingiae. A second 5·5‐kb cryptic plasmid pEPIR5, found in 12 E. piriflorinigrans strains, was also sequenced revealing mobilization and replication proteins with similarities to many small ColE1‐type plasmids in Erwinia spp. and other enterobacteria. Functional analyses of pEPIR37 introduced into a strain of E. amylovora cured of pEA29 plasmid, which has a reduced virulence, showed a role in increasing symptom development similar to that observed in E. amylovora carrying plasmid pEA29.     

Infection frequency of mature apple fruit with Erwinia amylovora deposited on pedicels and its survival in the fruit stored at low temperature

The infection frequency of mature apple fruit by Erwinia amylovora and the survival of E. amylovora in the fruit stored at low temperature were investigated. The fruit stems (pedicels) of 460 mature apple fruit were inoculated with 10⁵ or 10⁴ cfu of bioluminescent E. amylovora, tagged with lux genes. Nine days after inoculation, 43% and 27% of the fruit inoculated with 10⁵ and 10⁴ cfu, respectively, were infected. All infected fruit looked healthy. After 6 months of storage at 5°C, almost all of the 142 infected fruit had viable E. amylovora. Of the fruit containing E. amylovora internally, 19.5% had latent infections and the rest had blight symptoms. E. amylovora was not uniformly distributed in the fruit flesh, and internal brown lesions were observed where E. amylovora was densely distributed. These findings showed that mature apple fruit may be infected with E. amylovora, especially as latent infections, and act as a source for long-range dissemination.     

The undiscovered potential of dehydroproline as a fire blight control option

The non‐protein amino acid 3,4‐dehydro‐l ‐proline (DHP) significantly reduced the incidence of fire blight infection on immature pear fruits infected with wildtype Erwinia amylovora. DHP also inhibited biofilm formation in both streptomycin‐sensitive and ‐resistant strains of E. amylovora and induced dispersal of preformed biofilms in the streptomycin‐sensitive strain. The investigations shed light on the hitherto undiscovered ability of DHP to inhibit bacterial biofilms and its potential as a chemical control option for fire blight.     

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.     

Extended longevity of Erwinia amylovora vectored by honeybees under in vitro conditions and its capacity for dissemination

Fire blight outbreaks in Korea were first reported in 2015. Regular outbreaks have occurred since, indicating a continuous cycle of the fire blight pathogen in Korea. We determined the role of Apis mellifera (honeybee) as a vector of Erwinia amylovora by verifying the following: (a) E. amylovora longevity in/on honeybees; (b) the most common body parts that carry the bacteria; (c) the rate of bacterial spread to healthy host organs; and (d) the relationship between dispersal of viable but nonculturable (VBNC) and virulent bacterial cells. E. amylovora survived for 15 days on the exterior of honeybee bodies and was most abundant on the abdomen in comparison to other areas such as the labellum, wings, and hind legs. In the digestive system of honeybees, E. amylovora survived for 7 days, and bacteria were found in faeces for 3 days after exposure. The bacteria are likely to be VBNC on honeybees. Honeybees that were contaminated with bacteria transferred E. amylovora to healthy immature apple fruit, shoots, and flowers for 10 days after exposure. E. amylovora was also transferred from inoculated plant parts to uncontaminated honeybees. In addition, bacteria moved from inoculated plant tissues to unexposed honeybees and then from these honeybees to healthy plant tissues. Therefore, E. amylovora can survive in/on honeybees for extended amounts of time, which contradicts previous reports. The bacteria moved to host tissues via honeybees, suggesting that honeybees are the vectors of E. amylovora and play a role in the development of new outbreaks of fire blight disease in the central regions of Korea.     

Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 10⁶-6x 10⁷ CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 10³cfu ml^–1, which corresponds to 1cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.     

Alternative inoculum sources for fire blight: the potential role of fruit mummies and non‐host plants

Fire blight is the most damaging bacterial disease in apple production worldwide. Cankers and symptomless infected shoots are known as sites for the overwintering of Erwinia amylovora, subsequently providing primary inoculum for infection in the spring. In the present work, further potential sources of inoculum were investigated. Real‐time PCR assays covering a 3‐year‐period classified 19·9% of samples taken from fruit mummies as positive. Bacterial abundance in fruit mummies during autumn, winter and spring was up to 10⁹ cells per gram of tissue and correlated well with later infection rates of blossoms. Blossoms of non‐host plants growing close to infected trees were also shown to be colonized by E. amylovora and to enable epiphytic survival and propagation of bacteria. The results indicate a potential role of fruit mummies and buds in overwintering and as a source of primary inoculum for dissemination of the pathogen early in the growing season. Non‐host blossoms may also serve as an inoculum source in the build‐up of the pathogen population. Both aspects may contribute significantly to the epidemiology of E. amylovora. The significance of infected rootstocks as an inoculum source is also discussed. Fruit mummies might be used to determine pathogen pressure in an orchard before the beginning of the blooming period.     

Erwinia amylovora can pass through the abscission layer of fruit-bearing twigs and invade apple fruit during fruit maturation

Invasion of apple fruit by Erwinia amylovora from fruit-bearing twigs through the abscission layer at fruit maturation was examined. Erwinia amylovora (ca. 10⁵ cfu) tagged with bioluminescence genes from Vibrio fischeri was deposited in artificial wounds on fruit-bearing twigs of apple trees grown in a containment greenhouse on September 22, 27, or October 5, 2004. On October 22, 176 apples were harvested and cut horizontally in half. The upper halves were stamped on plates of selective medium, and the lower halves were flooded with iodine solution to assess maturity. All fruit were symptomless and fully mature. The pathogen was recovered from 19 (10.8%) apples. The result showed that if at least ca. 10⁵ cfu of E. amylovora are present in fruit-bearing twigs at the time of fruit maturation, the bacteria can pass through the abscission layer into the fruit, even though the mature fruit lack symptoms.     

Gene for pathogenicity and ability to cause the hypersensitive reaction cloned from Erwinia amylovora

A genomic library of Erwinia amylovora isolate T was constructed in the cosmid pLAFR3 and maintained in Escherichia coli. Clones were transferred individually by conjugation into the non-pathogenic isolate P66 of E. amylovora. Transconjugants were screened for restoration of pathogenicity to pear by stab inoculation into sections of immature pear fruits. Three clones complemented P66 restoring pathogenicity and ability to cause the hypersensitive reaction (HR) in Phaseolus vulgaris. Restriction mapping and hybridization experiments showed that the three clones had a common 3·7 kb fragment of E. amylovora DNA. Sub-cloning and insertion mutagenesis with Tn5-lac confirmed that a determinant of pathogenicity and ability to cause the HR (hrp gene) was located on a 2·1 kb HindIII/BamHI fragment within the common DNA. Hybridization experiments using the 2·1 kb HindIII/BamHI fragment as a probe demonstrated that the hrp gene was located in the chromosome of isolate T and that homologous sequences were present in the non-pathogenic isolates P66 and S. Clones which restored hrp function did not affect the growth of isolate P66 in minimal or nutrient-rich media. Transconjugants of Pseudomonas syringae pv. phaseolicola race 1 harbouring the hrp gene(s) cloned from E. amylovora did not cause the HR in susceptible cultivars of bean but symptoms developed more slowly than in the absence of the clones or with pLAFR3 alone.     

基于MaxEnt模型预测梨火疫病菌的潜在地理分布

为探究梨火疫病菌解淀粉欧文氏菌Erwinia amylovora在全球的潜在地理分布,基于其全球分布数据和筛选得到的环境变量,利用MaxEnt模型对其在当前气候和未来气候条件下的潜在地理分布进行预测,并利用刀切法和皮尔逊相关性分析法筛选对梨火疫病菌分布有重要影响的环境变量。结果显示,对梨火疫病菌分布有重要影响的环境变量包括2月平均最高温度、1月平均降水量、7月平均最低温度、温度变化方差、昼夜温差月均值和7月平均降水量,表明春季和夏季的温度和降水对梨火疫病菌的分布有较大影响。在当前气候条件下,梨火疫病菌在全球的适生区分布较广,适生区总面积达到5.58×10⁷ km²,且高适生区主要分布在北美洲沿海地区、地中海沿岸和亚洲中部及东部的部分地区;梨火疫病菌在我国的适生区总面积为7.36×10⁶ km²,占全国陆地总面积的76.70%;在未来气候SSP126和SSP585情景下,梨火疫病菌在全球的适生区总面积分别为5.52×10⁷ km²和5.24×10⁷ km²。表明梨火疫病菌对我国大部分地区有潜在威胁,应加强监测与防控。     

Erwinia amylovora populations resistant to oxolinic acid in Israel: prevalence, persistence and fitness

Oxolinic acid (OA) has been the only bactericide used against fire blight in pear and quince orchards in Israel since 1998. OA-resistant Erwinia amylovora strains (Ea-OA^R) were detected in several orchards in two restricted areas in the northern Galilee region during 1999–2001. In the following years, resistant strains could not be detected in some of these locations. Documenting the fate of Ea-OA^R strains in commercial orchards at eight sites in northern Israel during 2000/03 revealed that the resistant population appeared irrespective of the number of sprays applied and the severity of the disease. The persistence of the Ea-OA^R populations varied from site to site, ranging from 4 to 20 months; these differences could be attributed to the fire blight management activities of growers. Comparative studies on the fitness of Ea-OA^R and E. amylovora strains sensitive to OA (Ea-OA^S) were conducted in vitro and in planta using two strains of each group. In four of the six comparisons, disease incidence on detached blossoms inoculated with Ea-OA^S was significantly higher than that on blossoms inoculated with Ea-OA^R. In two experiments conducted on 8-year-old pear trees grown under netting, the colonization of Ea-OA^S in blossoms, annual shoots and perennial spurs was significantly higher than that of the Ea-OA^R. In two experiments conducted on 2-year-old trees grown under netting in an experimental station, the incidence of shoots exhibiting fire blight symptoms and the rate of symptom progress within the branches were significantly higher in trees inoculated with Ea-OA^S than in those inoculated with Ea-OA^R. The results of this study suggest that OA-resistant E. amylovora strains have lower fitness than wild-type strains. These findings may have implications for fire blight management.     

Characterization of the host range and sensitivity to fungicides of Trichothecium spp. associated with fruit rot in the field and in storage

During 2017–2019, we observed Trichothecium spp. causing fruit rot in the field and in storage. This study was conducted to examine morphological differences of the species from different hosts, reveal the species’ potential host range, and evaluate the efficacy of five fungicides. Six strains of Trichothecium spp. isolated from nectarine, peach and walnut were selected. Although the colony morphology, mycelial growth rate and spore size differed among hosts, phylogenetic analysis based on the internal transcribed spacer and part of the 5ʹ end of the β-tubulin gene showed that all tested strains belonged to Trichothecium roseum. For its host range, 23 kinds of fruit were examined using T. roseum strain YT-1 as an inoculum; 10 kinds of fruit, including pear, apple, mango, Chinese chestnut, pepino melon, fig and durian, were susceptible to T. roseum, with minimum inoculation concentrations ranging from 10⁴ to 10⁵ spores/ml. The fungicides that most effectively inhibited the six isolates were fluazinam and fludioxonil, with EC₅₀ values of 0.07–0.1 and 0.01–0.04 μg/ml, respectively, followed by difenoconazole (0.81–2.96 μg/ml), boscalid (5.43–13.51 μg/ml) and azoxystrobin (9.18–27.25 μg/ml). Improvement of the shelf life of nectarines held in plastic trays was explored using allyl isothiocyanate (AITC) against T. roseum YT-1. The application of 10 μl/L AITC significantly suppressed disease symptoms. The findings provide useful information for future disease emergency management in the field and for food preservation.     

Identification of dspEF, hrpW, and hrpN loci and characterization of the hrpN Ep gene in Erwinia pyrifoliae

Erwinia pyrifoliae, the causal pathogen of shoot blight in the Asian pear tree (Pyrus pyrifolia cv. Singo), is host-specific and endemic to Korea. To identify the genes associated with the hypersensitive response (HR) and pathogenicity, a genomic library of E. pyrifoliae WT3 was constructed, and the cosmid clone Escherichia coli (pCEP33) was selected. Sequence analysis of 19.7-kb pCEP33 determined disease-specific (dsp) region homolog and approximately 40% of the hrp genes, which included hrpW, hrpN_Ep, hrpV, hrpT, hrcC, hrpG, hrpF, and partial hrpE homologs, with respect to the cluster of Erwinia amylovora. Additionally, two open reading frames, ORFD and ORFE, were found downstream of the dspEF region. The results of the sequence analysis showed that the pCEP33 did not contain any hrp regulatory genes or most of the genes encoding components of the Hrp protein secretion system. The hrpN_Ep gene of E. pyrifoliae contained five intergenic nucleotide fragment insertions (INFIs) and produced the HR elicitor protein harpin_Ep, with a molecular mass of approximately 44kDa. The purified HrpN_Ep protein elicited faster and stronger HR when infiltrated into tobacco leaves than did HrpN_Ea from E. amylovora. To observe the role of the hrpL gene in the expression of HrpN_Ep, the pEL2 containing hrpL was used to transform E. coli (pCEP33). Expression of HrpN_Ep in E. coli (pCEP33 + pEPL2) was detected with an immunoblot using antiserum raised against HrpN_Ep, indicating a role of hrpL gene in enhancing the expression of HrpN_Ep.     

Note: Evaluation of antibacterial activity of essential oil of<Emphasis Type="Italic">Rosa damascena</Emphasis> on<Emphasis Type="Italic">Erwinia amylovora</Emphasis>

Significant antibacterial activity was observed in the essential oil (E.O.) ofRosa damascena Mill. and the Minimum Bactericidal Concentration (MBC) of the E.O. was determined as 1386.5 μg ml⁻¹ forErwinia amylovora, the causal agent of fire blight disease. Ooze formation on immature pears and lesion formation in artificially inoculated shoots were completely (100%) prevented by the essential oil ofR. damascena (1500 μg ml⁻¹), essential oil ofThymbra spicata var.spicata (500 μg ml⁻¹) and streptomycin (100 μg ml⁻¹). Copper oxychloride plus maneb significantly reduced ooze formation and lesion formation, but the control was less than that obtained with the essential oils or streptomycin. The essential oil ofR. damascena may be a useful natural bactericide for the control of the fire blight pathogen,E. amylovora. http://www.phytoparasitica.org posting July 14, 2004.     

Characterization of the incompatible interaction between Erwinia tracheiphila and non-host tobacco (Nicotiana tabacum)

Bacterial wilt is one of the most destructive diseases affecting a wide range of crops in the Cucurbits family including muskmelon (Cucumis melo), cucumber (Cucumis sativus), and squash (Cucubita pepo). The disease is caused by Erwinia tracheiphila, a Gram-negative and xylem-inhabiting species of Erwinia, which pathogenic mechanism is poorly understood. Many Gram-negative phytobacteria induce hypersensitive response (HR) in non-host plants, an immunity reaction triggered by pathogen recognition. With some exemptions, Erwinia species—notably E. amylovora, the causative agent of fire blight of rosaceous crops, and the reclassified soft rot pathogens, Pectobacterium and Dickeya species (formerly E. carotovora and E. chrysanthemi)—have been known to elicit HR in tobacco. However, concerning its pathogenic mechanism, the elicitation of classic HR has not been reported for some less-studied Erwinia species including E. tracheiphila. We characterized the induction of HR by the bacterial wilt pathogen in tobacco (Nicotiana tabacum cultivar ‘Xanthi’) using visual and physiological methods. We surveyed 21 E. tracheiphila strains and found that all of them elicited programmed cell death. Three strains (HCa1-5, UnisCu1-1, and MISpSq) fluorescently labeled with GFP could be visualized in the infiltrated leaves. We aligned the sequences of their HR-inducing protein, harpin (HrpN), predicted the secondary structures, and located the position of putative HR elicitors. We discovered differences between Cucurbita and Cucumis strains and found a close association of E. tracheiphila HrpN with those of Pantoea sp., Erwinia piriflorinigrans, and Erwinia pyrifoliae. Pre-infiltration of tobacco leaves with a lower cell population prevented HR following a subsequent challenge at the same area with HR-inducing levels of inoculum. The selected strains induced leaf conductivity levels similar to the HR-inducing E. amylovora strain E9, and their populations in the leaves decreased days after infiltration. Our results indicate that E. tracheiphila induces a classic HR in tobacco just like other HR-inducing Erwinia species.