Erwinia amylovora hrpN mutants,blocked in harpin synthesis,express a reduced virulence on host plants and elicit variable hypersensitive reactions on tobacco

Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting many rosaceous plants and especially pear tree and apple tree. A protein named harpin, secreted through the Hrp secretion pathway and able to elicit an hypersensitive reaction (HR) on tobacco has recently been isolated. Mutants inhrpN, the gene encoding harpin were described as non pathogenic on immature pear fruit and unable to elicit an HR on tobacco [Weiet al., 1992; Wei and Beer, 1993]. In this paper, the phenotype on plant ofhrpN mutants was carefully determined.hrpN mutants expressed a weak but significant virulence on host plants. Furthermore, when infiltrated into tobacco leaf mesophyll, thehrpN mutants elicited varied responses that fluctuated from null reaction to full necrosis of the infiltrated area. These results show that harpin is not absolutely required neither for pathogenicity on host plant nor for elicitation of an hypersensitive reaction on tobacco. Furthermore, in all the tests performed, mutant blocked in harpin secretion remained non pathogenic and unable to elicit an HR on tobacco. This suggests that factor(s), different from harpin, involved both in pathogenicity and HR eliciting ability is (are) secreted through the Hrp secretion pathway.Abbreviations HR hypersensitive reaction - NSI necrosis severity index - CFU colonie forming units     

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.     

Effects of pear tree physiology on fire blight progression in perennial branches and on expression of pathogenicity genes in Erwinia amylovora

The interaction between Erwinia amylovora (the causal agent of fire blight) and the physiological status of pear trees was examined under orchard conditions. The physiological status of the trees was defined qualitatively, using host phenology and vigour as measures, and quantitatively, using the sorbitol content in annual shoots as a measure. Qualitatively, tree response to fire blight was governed by phenological stage at the time of infection and vigour: low vigour trees inoculated in the autumn (just before entering dormancy) and high vigour trees inoculated in the spring (soon after bloom) were more susceptible than high vigour trees inoculated in the autumn and low vigour trees inoculated in the spring. Quantitatively, the rate of symptom progression in perennial branches (SPR) was significantly (P ≤ 0.001) correlated to the absolute value of the rate of sorbitol content change (|SCR|). The relationship between hrp genes expression of transformed E. amylovora (estimated according to hrpE and hrpJ expression) and |SCR| was determined on 1 year-old trees. Expression of hrp genes was significantly correlated with |SCR| (P = 0.004) and 63.5% of the variability in the hrp genes expression was attributed to |SCR| values. The expression of hrp genes increased gradually and asymptotically with increasing |SCR| values; further increase in |SCR| did not affect the expression.     

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.     

Characterisation of naturally occurring <Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains lacking the common plasmid pEA29 and their detection with real-time PCR

Erwinia amylovora, the causal agent of fire blight, carries the common plasmid pEA29 of 29 kb. To screen for occurrence of natural strains without plasmid pEA29, we applied PCR analysis with primers from the plasmid and the chromosomal ams region. In addition, a described TaqMan probe from pEA29 and newly designed primers from the ams-region were used for identification by real-time PCR. One strain isolated in Iran, one strain from Spain and two strains from Egypt lacked plasmid pEA29. From a recent screening series in southern Germany, in 123 E. amylovora strains from necrotic fire blight host plants, one strain was found without the common plasmid. The strains without pEA29 were virulent in assays with immature pears and on apple seedlings, but showed a reduced growth level in minimal medium without amino acids and thiamine. Transposon-labelled pEA29 was transformed into the plasmid-free strains resulting in restoration of this growth deficiency. The plasmid was stably maintained in these E. amylovora cells. The newly designed chromosomal primers for conventional and for real-time PCR identified E. amylovora strains in field samples lacking pEA29. These variants are apparently rare, but were detected in isolates from different regions in the world with fire blight.     

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.     

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

The Gram‐negative bacterium Erwinia amylovora, causal agent of fire blight disease in pome fruit trees, encodes a type three secretion system (T3SS) that translocates effector proteins into plant cells that collectively function to suppress host defences and enable pathogenesis. Until now, there has only been limited knowledge about the interaction of effector proteins and host resistance presented in several wild Malus species. This study tested disease responses in several Malus wild species with a set of effector deletion mutant strains and several highly virulent E. amylovora strains, which are assumed to influence the host resistance response of fire blight‐resistant Malus species. The findings confirm earlier studies that deletion of the T3SS abolished virulence of the pathogen. Furthermore, a new gene‐for‐gene relationship was established between the effector protein Eop1 and the fire blight resistant ornamental apple cultivar Evereste and the wild species Malus floribunda 821. The results presented here provide new insights into the host–pathogen interactions between Malus sp. and E. amylovora.     

Recent Progress in Breeding for Fireblight Resistance in Apples and Pears in North America1

Resistance in apple is evaluated by needle–inoculation of succulent shoot tips with 10⁶–10⁷ cells of a virulent isolate of Erwinia amylovora (Burr.) Winslow et al. (the incitant of fireblight) and measurement of the resulting cortical lesion when extension is complete. Data are now available on practically all commercial cultivars, some of which have a useful level of resistance. Some newer cultivars, particularly those with resistance to scab (Venluria inaequalis [Cooke] Wint.) derived from Malus floribunda, have good resistance to E. amylovora. A very high level of resistance is present in Asiatic Malus species, including M. x robusta, M. x sublo–bata, M. x atrosanguinea, and M. prunifolia, and in the North American species M. fusca. This type of resistance appears to be inherited polygenically, and because of its detectability in young seedlings can be used conveniently in breeding. Objectives of pear breeding programs are aimed at developing superior fruit quality combined with resistance to fireblight, psylla, and Fabraea leaf spot. Many high quality cultivars of Pyrus communis are extremely susceptible to E. amylovora and sensitivity appears to be controlled by a dominant gene Se. A high level of resistance is present in P. ussuriensis but varies considerably between clonal selections of other Pyrus species. Pear seedlings from controlled pollinations are artificially inoculated in the glasshouse with a similar bacterial suspension as used for apples, and only the most resistant ones are selected and planted in the field for future evaluation. In Beltsville, heritability studies of crosses between non–sensitive parents have indicated that selection for resistance within progenies results in a high degree of genetic gain. Interspecific hybridization has an advantage over P. communis crosses mainly when insect or fungus resistance is required.     

Citrus black spot intensity and yield losses on sweet orange are affected by Phyllosticta citricarpa inoculum concentration and fruit developmental stage

Citrus black spot (CBS) symptom expression on sweet oranges has been reported to be affected by the fruit developmental stage and inoculum concentration in greenhouse conditions. However, there is little information concerning the period in which the fruit is susceptible in commercial orchards. This study assessed the influence of fruit age and inoculum concentration of Phyllosticta citricarpa on CBS intensity and fruit drop in the field. Two field trials were conducted in commercial ‘Valencia’ orchards in São Paulo state, Brazil. Fruit were inoculated from October (petal fall stage) to July (c.6.5 cm diameter), with 10¹, 10³ and 10⁵ pycnidiospores/ml. CBS symptoms and fruit drop were observed in higher levels for fruit inoculated from October to February than from March to July. In both trials, when fruit were inoculated 10 times from October to July or single-inoculated in November or December, high CBS severities of 11%–22% were observed and the proportion of fruit remaining on the tree was 48%–77%; in contrast, noninoculated fruit had severity below 1.1% and 90% remained on the tree. Inoculations in November or December of green fruit with 1.5–3.0 cm diameter using 10⁵ pycnidiospores/ml were associated with higher CBS intensity and fruit drop and shorter incubation period than inoculations with lower concentrations. This work provides a better understanding of fruit stages and P. citricarpa inoculum concentrations most related to symptom expression and losses under natural conditions and may be helpful for improvement of CBS management in the São Paulo citrus belt.     

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.     

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.     

Necrotrophic behaviour of Erwinia amylovora in apple and tobacco leaf tissue

An important issue related to the epidemiology of fire blight, a devastating disease of apples and pears, is how its causal agent, the bacterium Erwinia amylovora, survives and disseminates in the environment. Almost no information is available on the possibility of this pathogen overwintering as a necrotroph. In this study, bacterial survival in dead apple and tobacco (a non‐host) leaf tissues was addressed. In necrotized leaves collected 5, 6, 7 and 8 months following shoot inoculation of apple trees, viable E. amylovora cells were present in over 50% of samples from the midrib and in over 10% of samples from lateral veins, but were never found in parenchyma. Using a PCR‐based method, pathogen DNA was detected in more than 50% of samples that were found to be free of viable cells by conventional plating out. However, PCR analysis was insufficient to distinguish between the DNA of viable and dead bacteria. Sugars appropriate for bacterial growth were found in dead apple leaves. In spot‐inoculated attached apple and tobacco leaves, a remarkable increase in the bacterial population was observed in lesions that developed as a hypersensitive response (HR). As in other necrotrophic interactions, bacterial proliferation was associated with massive hydrogen peroxide production and progression toward plant cell death. The results indicate that E. amylovora has an ability to survive as a semi‐necrotroph or necrotroph, which allows for overwintering in dead apple leaves.     

Virulence, resistance to magainin II, and expression of pectate lyase are controlled by the PhoP-PhoQ two-component regulatory system responding to pH and magnesium in Erwinia chrysanthemi 3937

Marker-exchanged mutants of phoP and phoQ of Erwinia chrysanthemi (Ech) strain 3937 became more sensitive to the cationic antimicrobial peptide (CAMP) magainin II than did the wild type at a low Mg²⁺ concentration and at either acidic or neutral pH. At high Mg²⁺ and acidic pH, only the phoQ mutant, but not the phoP mutant, became more sensitive to magainin II than did the wild type; both mutants were more sensitive at neutral pH. The hyperinduction of Pel synthesis in medium containing plant extracts and polygalacturonic acid (PGA) was confirmed in the wild type but not in the mutants at low Mg²⁺ and neutral pH. However, Pel was hyperinduced at high Mg²⁺ and neutral pH in these mutants but not in the wild type. Maceration was also greatly reduced by these mutants compared to the wild type when the inoculum was precultured and then resuspended in the medium with low Mg²⁺ at neutral pH. However, when bacteria were precultured and resuspended in the medium with high Mg²⁺ at neutral pH, severe maceration was observed in these mutants but not in the wild type. Thus, at low Mg²⁺, PhoP-PhoQ TCS seems to be stimulated for maceration and the hyperinduction of Pel synthesis. At high Mg²⁺, however, PhoP-PhoQ TCS may be repressed for these phenotypes, and PhoP may be controlled by a mechanism(s) other than PhoQ regulation.     

An assessment of the relative resistance of three hawthorn species to three strains of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> using three different inoculation methods

Fire blight is an important disease of hawthorn plants. In this study, the level of susceptibility of three hawthorn species (Crataegus monogyna, Crataegus laevigata, Crataegus persimilis) to the bacterium Erwinia amylovora was investigated. The results showed that all species were highly susceptible to this pathogen. In addition, the relative virulence of three different E. amylovora strains on the above species was examined. Variability among the strains was found, with strain 3 being the most virulent and strain 1 the least.     

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.     

Het toetsen van stammen vanNectria cinnabarina (Tode) fr. op levende takken in vitro

Summary Some types of elm hybrids resistent to the Dutch elm disease turned out to be susceptible toNectria cinnabarina (Tode) Fr. This fungus may behave as a parasite, killing branches. Virulent and non-virulent strains are known. A method is described for cultivatingNectria-strains in vitro on living desinfected branches ofAcer and ofUlmus. Three weeks after inoculation with a virulent strain the branches were killed. They were covered all over by a thick layer of mycelium. The mycelium of non-virulent strains could not kill the branches. It developed only sparsely. By this method it will be possible to select the most virulent strains out of a collection of isolates in order to use them for testing elm hybrids on their resistence toNectria cinnabarina.     

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.