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.     

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.     

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.     

Efficacy of oxolinic acid and other bactericides in suppression ofErwinia amylovora in pear orchards in Israel

The efficacy of oxolinic acid (at 200 and 300 μg a.i./l) and of several antibiotic compounds (streptomycin sulfate at 100 μg a.i./l, glycocide B at 700 μg a.i./l, kasugamycin at 80 μg a.i./l and gentamicin sulfate at 30 and 60 μg a.i./l) againstErwinia amylovora, the causal agent of fire blight in pears, was evaluated in 43 orchard experiments in 1997–2000 in Israel. In addition to the above orchard experiments, the efficacy of the bactericides was tested in live experiments with artificial inoculation. Natural fire blight symptoms were observed in 16 of the 43 experiments; in 13 of them, disease intensity and its distribution among the experimental plots provided a basis for data analysis, leading to reliable conclusions concerning the efficacy of the tested bactericides. Oxolinic acid at 300 μg a.i./l was highly effective againstE. amylovora and reduced disease severity significantly in all experiments, as compared with the untreated plots; however, a concentration of 200 μg a.i./l was not effective in some cases. Among the tested antibiotics, only gentamicin sulfate was as effective as oxolinic acid. Results of the artificial inoculation experiments corroborated those obtained in the naturally infected orchards. The pre-infection activity of oxolinic acid was determined on blossom clusters that were sprayed with the bactericide before inoculation. Control efficacy on blossom clusters sprayed 1–4 days before inoculation ranged from 68% to 80%, a level which did not differ significantly from that observed on blossom clusters sprayed on the day of inoculation (80% control). The postinfection activity of oxolinic acid was determined on blossom clusters that were sprayed with the compound after inoculation. Oxolinic acid was as effective when applied 1 or 2 days after inoculation as when it was applied on the day of inoculation; however, application of the bactericide 3 days after inoculation no longer resulted in significant disease suppression. Oxolinic acid has been used commercially in Israel since 1998 with appreciable success. Contribution No. 533-00 from the Agricultural Research Organization     

Behavior of <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with bioluminescence genes on rice plants

Erwinia ananas, the causal agent of bacterial palea browning of rice, was transformed with bioluminescence genes to clarify their behavior on rice plants. Transformant CTB009T2 was used to inoculate rice plants, and the subsequent bioluminescence of CTB009T2 was observed using a two-dimensional luminometer. Luminous spots frequently appeared on anthers after flowering and on dead tissues such as leaf tips, lower leaf sheaths, and leaf blades. In spikelets that developed the disease symptom on the palea, luminous spots appeared 48h after flowering on stigmas, basal parts of ovaries, and lodicules. These results indicate that postflowering anthers and dead tissues on rice plants are important sites for a rapid increase in the pathogen population, and that the multiplication of the pathogen on internal tissues of spikelets after flowering is associated with the appearance of browning.     

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     

Use of a diagnostic medium for<Emphasis Type="Italic">in situ</Emphasis> determination of the response of<Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains to bactericides

Erwinia amylovora, the causal agent of fire blight, is managed by application of bactericides to protect fruit tree blossoms from infection. Monitoring the response ofE. amylovora strains to bactericides is crucial for adequate disease management. The coliform agar medium produced by Merck was recently reported as an effective tool for rapid diagnosis ofE. amylovora (RD-medium). The objective of the present study was to examine the possibility of using the RD-medium forin situ determination of the response ofE. amylovora strains to oxolinic acid and streptomycin. The phenotypic response of 48E. amylovora strains isolated in 2002 to both bactericides was determined with the RD-medium and, for comparison, by a routine laboratory test. The results of 45 samples (93.7%) were in agreement with the findings of the routine laboratory test. Aχ ² test rejected the null hypothesis that the phenotypic characteristics as determined by the two respective methods differed significantly (P=0.389). Thein situ test was implemented on a national scale in 2003 and the results were in agreement with those obtained in laboratory tests, which suggests that this medium can be usedin situ for monitoring the appearance of resistance inE. amylovora populations. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Confidence intervals for the estimation of the incubation period of fire blight following Billing's prediction system 1

An equation of Billing's system 1 permits an estimate of the length of the incubation period (I) of fire blight (Erwinia amylovora) in rosaceous plants. The confidence interval of I was determined using Billing's own data (90% confidence interval: α6.3 days). By transformation of I, the confidence interval could be reduced (α3.5 days). Temperature was more important than rain with respect to the rate of development of fire blight. The effects of temperature and rain on the rate of development showed a strongly positive interaction.Samenvatting De lengte van de incubatieperiode van bacterievuur (Erwinia amylovora) kan geschat worden met een formule uit Billings voorspellingssysteem 1. Om enig inzicht te verkrijgen in de nauwkeurigheid van Billings systeem, werd het betrouwbaarheidsinterval van de geschatte incubatieperiode berekend aan de hand van Billings eigen proefgegevens. Bij een betrouwbaarheid van 90% werd een afwijking van 6,3 dagen gevonden. Het betrouwbaarheidsinterval kon verkleind worden door middel van transformatie van de regressieparameters en toepassing van meervoudige regressie (bij 90% betrouwbaarheid een afwijking van 3,5 dagen).De temperatuur had meer invloed op de ontwikkelingssnelheid van bacterievuur dan regen. Verder vertoonden temperatuur en regen een sterk positief interactief effect op de ontwikkelingssnelheid.     

The interacting effects of temperature,duration of wetness and inoculum size on the infection of pear blossoms by<Emphasis Type="Italic">Erwinia amylovora</Emphasis>, the causal agent of fire blight

Pear blossoms are the plant tissue that is most vulnerable to infection byErwinia amylovora (Burrill) Winslowet al., the causal agent of fire blight. The interacting effects of temperature, wetness duration and inoculum size on the development of fire blight symptoms in detached pear blossoms were determined in three sets of experiments conducted under controlled conditions. It was expected that this information would facilitate the improvement of a warning system used in fire blight management. Results of the ANOVA tests of the data revealed highly significant interactions among the factors tested. The factors that contributed most to disease incidence were temperature and inoculum size; effects of wetness duration were significant in some cases, but that effect was small. It was further demonstrated that the effects of the interaction of these factors on the incidence of blossom infection may be understood in terms of the general concept of compensation. According to this concept, conditions highly favorable for one of the factors essential for pathogen development may compensate for other factors, for which the conditions are less favorable. As a result of the complex interactions observed between the biotic and abiotic factors, because of compensation relationships and because some of the factors cannot be estimated adequately (for example, inoculum level), it was concluded that it is not yet possible to improve fire blight management by using data on the quantitative relationships between biotic and abiotic factors. Contribution No. 532/02 from the Agricultural Research Organization. http://www.phytoparasitica.org posting Feb. 20, 2003.     

Enhanced colonization and pathogenicity of Erwinia amylovora strains transformed with the near‐ubiquitous pEA29 plasmid on pear and apple

Three plasmid‐free strains of Erwinia amylovora, the causal agent of fire blight disease of pome trees, one from Iran, one from Egypt and one from Spain, were transformed with the near‐ubiquitous nonconjugative pEA29 plasmid from a wild‐type strain and characterized. The plasmid‐deficient strains were levan‐ and slime‐positive, motile, chemotaxis‐positive, induced HR on Nicotiana tabacum var. xanthi but produced several‐fold less amylovoran and were weakly pathogenic on pear slices and apple seedlings compared to plasmid‐bearing wild‐type strains. When inoculated onto wounded young apple (cv. Royal Gala) leaves, the plasmid‐free strains labelled with green fluorescent protein gene (gfp) were mainly restricted to the inoculation site at the leaf tips, in contrast to the plasmid‐carrying wild‐type strains that moved into the midrib xylem vessel and colonized the adjacent parenchyma cells. Upon introduction of the transposon‐labelled pEA29 plasmid, amylovoran production, degree of oozing and tissue necrosis on pear slices were significantly elevated in all three strains, whilst the levels of levan and levansucrase declined. Only the strains from Iran and Egypt gained the ability to invade and colonize the young apple leaves following the introduction of pEA29. It is concluded that acquisition of the nonconjugative near‐ubiquitous plasmid may not necessarily confer increasing pathogenicity in all bacterial strains.     

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.     

An oligonucleotide probe for the detection of Erwinia herbicola and Erwinia ananas

An oligonucleotide probe targeting the rRNA of Erwinia herbicola and Erwinia ananas was designed to detect their cells using fluorescence in situ hybridization. The Cy3-labeled probe hybridized strongly with these species but very weakly with nontarget species such as Erwinia mallotivora, Erwinia nigrifluens, Erwinia cypripedii, Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. This technique visualized E. herbicola cells after inoculation of kumquat fruits. The probe is promising as a tool for studying population dynamics of E. herbicola and E. ananas.     

Mutations in ams genes of Erwinia amylovora affect the interactions with host plants

A large region for exopolysaccharide (EPS) production that is involved in the pathogenicity of Erwinia amylovora strain CFBP1430, cloned previously, was further characterized in this study. An 8.4 Kb-long sub-cloned fragment from that ams (amylovoran synthesis) region was mutagenized in vitro by Tn3Gus insertion. Several new chromosomal mutants were obtained by marker exchange. The phenotype of those mutants was determined with regard to pathogenicity, EPS production, and growth in planta. It appeared that alteration in EPS synthesis was correlated with altered pathogenicity and poor (or no) growth in planta. Altogether these data argue for a direct role of EPS in bacterial multiplication in planta, and/or for an indirect role by protecting the bacteria against host defence reactions.     

Identification of potential virulence genes in Erwinia chrysanthemi 3937: transposon insertion into plant-upregulated genes

Erwinia chrysanthemi 3937 is a soft-rotting plant pathogen in Enterobacteriaceae. It attacks a wide range of plant host species. Previously, we identified dozens of E. chrysanthemi 3937 genes induced during plant infection by microarray differential display. Here, we have mutated plant-upregulated and putatively plant-upregulated genes in E. chrysanthemi 3937 using a transposon insertion method. Of 57 mutants produced, 8 were significantly reduced in maceration in African violet leaves. These 8 E. chrysanthemi genes are similar to Escherichia coli purU (formyltetrahydrofolate deformylase; ASAP20623) and wcaJ (undecaprenylphosphate glucosephosphotransferase; ASAP18556), Bacillus subtilis dltA (d-alanine-d-alanyl carrier protein ligase; ASAP19406), Pseudomonas syringae PSPTO2912 (ABC transporter, periplasmic glutamine-binding protein; ASAP15639), Pseudomonas aeruginosa pheC (cyclohexadienyl dehydratase; ASAP19773), P. syringae syrE (peptide synthase; ASAP19989), Vibrio vulnificus VV12303 (unknown protein; ASAP18555), and Yersinia pestis speD (S-adenosylmethionine decarboxylase; ASAP20536). In some of the genes, possible roles in virulence could be postulated based on the functions of their homologues. This work demonstrated that a low proportion of pathogenicity-related genes were among the plant-upregulated genes of E. chrysanthemi 3937. This study and further dissection of these putative virulence genes should lead to new insights into infection mechanisms in pathogens.     

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.     

A revision of Billing's potential doublings table for fire blight prediction

In her fire blight prediction systems, E. Billing (1978, in: P.R. Scott & A. Bainbridge (Eds), Plant disease epidemiology, p. 159–166) has used the parameter potential doublings per day (PD) of the fire blight causing bacterium,Erwinia amylovora. Reconsideration of her calculations of PD revealed, however, that the PD values in Billing's table were underestimated. This leads to overestimation of the duration of incubation periods. A corrected PD table is presented. Sensitivity analyses indicated that day-of-the-year and latitude have little effect on the values in the PD table.Samenvatting Billing maakte in haar systemen waarin de ontwikkeling van bacterievuur wordt voor-speld, gebruik van de parameter potentiële verdubbelingen per dag (=PD) van de bacterievuur veroorzakende bacterieErwinia amylovora. Uit herberekeningen blijkt echter, dat de PD-waarden in Billings tabel onderschat worden. PD-onderschattingen leiden in haar systemen tot overschattingen van de duur van incubatieperioden. Een gecorrigeerde PD-tabel werd samengesteld. Uit gevoeligheidsanalyses bleek dat de dag van het jaar en de breedtegraad een gering effect hebben op de waarden in de PD-tabel.     

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.