Shulamit Manulis

Indole-3-acetic acid biosynthetic pathways in Erwinia herbicola in relation to pathogenicity on Gypsophila paniculata☆

Pathogenic strains of Erwinia herbicola incite crown and root galls in the flowering ornamental gypsophila. Both pathogenic and non-pathogenic strains of the bacterium readily produce indole-3-acetic acid in culture. Two pathways for biosynthesis of indole-3-acetic acid were identified in E. herbicola: (1) the indole-3-acetamide route occurs via the following reactions: l-tryptophan → indole-3-acetamide → indole-3-acetic acid, and (2) the indole-3-pyruvate route involves the following reactions: l-tryptophan → indole-3-pyruvate → indole-3-acetaldehyde → indole-3-acetic acid. Production of indole-3-ethanol was also linked to the latter pathway. Evidence for the existence of the two pathways was based on: (a) chemical identification of the respective indole intermediates by thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectroscopy; (b) production of indole-3-acetic acid by bacterial cells treated with the various indole intermediates; and (c) incorporation of 3-14C-l-tryptophan into the indole intermediates of the two pathways. In contrast to the indole-3-pyruvate pathway which was detected in all the pathogenic and non-pathogenic strains examined, the indole-3-acetamide pathway was detected only in the pathogenic strains of E. herbicola. The possible relationship between the indole-3-acetamide pathway and gall formation by E. herbicola is discussed.

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.

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.

Evaluation of local and imported fire blight warning systems in Israel.

ABSTRACT The possibility of using local and imported warning systems for the management of fire blight (caused by the bacterium Erwinia amylovora) in pears was tested in Israel from 1997 to 2000. Three imported systems (MARYBLYT 4.3, BIS95, and Cougarblight 98C) and one local system (Fire Blight Control Advisory [FBCA]) were used. All systems were tested in simulation experiments; MARYBLYT 4.3 and FBCA were also tested in orchard experiments under natural infections. Simulation experiments included 193 orchard-plots in which the time of disease onset enabled us to determine the date of infection. Thirty-five experiments were conducted in commercial orchards; in 10 of these, fire blight developed naturally. The performance of the imported warning systems was too variable to be accurately used under Israeli conditions. In the simulation experiments, the success rate (i.e., the capacity of the systems to predict the exact date of the occurrence of infection episodes) of the imported systems was low (3 to 55%) with considerably large variability among years (CV = 30 to 67%). Similar results were obtained in the orchard experiments for MARYBLYT 4.3: in only two of five experiments where plots were managed according to that system was disease severity significantly lower than that recorded in untreated control plots. In comparison, the local system, FBCA, predicted most infection episodes in the simulation experiments with low variability (99%, CV = 1.0%). In the orchard experiments, adequate disease suppression was achieved in all eight experiments in which FBCA recommendations were followed. We concluded that it was not possible to import and successfully implement fire blight warning systems in Israel that have been developed in regions with dissimilar environmental conditions.

CHARACTERIZATION OF THE AUXIN SYNTHESIS GENES OF ERWINIA HERBICOLA PV. GYPSOPHILAE

We present the DNA sequence of some 3590 base pairs from the virulence plasmid of the crown-gall-forming bacterium Erwinia herbicola pv. gypsophilae strain PD713. This region includes the entire transcription unit of an operon encompassing the IAA synthesis genes. These genes, designated iaaM and iaaH, reside on a 78-MDa native plasmid. The sequence analysis indicates that the operon contains two open reading frames of 562 (iaaM) and 460 amino acids (iaaH), corresponding to proteins with molecular weight of 62,473 and 49,300 daltons, respectively. When cloned and expressed in minicell-producing E. coli, this sequence encodes production of two proteins, 49 and 62 kDa. Both genes show moderate to high homology to IAA synthesis genes from other plant-associated bacteria. Analysis by primer extension of the transcription initiation site located it 63 bases upstream of the translation initiation codon.

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 1xa0year-old trees. Expression of hrp genes was significantly correlated with |SCR| (Pxa0=xa00.004) 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.

Detection of Erwinia herbicola pv. gypsophilae in gypsophila plants by PCR

Three PCR primer pairs, based on the cytokinins (etz) or IAA biosynthetic genes, were used for detecting Erwinia herbicola pv. gypsophilae in Gypsophila paniculata plants. The primers were specific to all gall-forming E. herbicola strains and distinguished them from saprophytic strains associated with gypsophila plants or from other gall-forming bacteria. In pure culture of the pathogen, less than one bacterial cell was detected with nested PCR using the etz primers - an increase of 100-fold in sensitivity as compared with single-round PCR. In the presence of plant extract a reduction of tenfold in sensitivity was observed by nested PCR. When cells were grown on a semi-selective medium prior to PCR (Bio-PCR), five cells from pure culture of the pathogen were detected. The bacteria could be detected by nested-PCR or Bio-PCR in symptomless gypsophila cuttings after 7 days. The Bio-PCR procedure described in this study can be used to establish disease-free nuclear stock of mother plants of gypsophila.

Testing a Rapid Diagnostic Medium for Erwinia amylovora and Development of a Procedure for Sampling Blossoms in Pear Orchards

ABSTRACT The coliform agar produced by Merck was tested for rapid diagnosis of Erwinia amylovora (the causal agent of fire blight) in pear blossoms. The medium enabled the diagnosis to be completed within 36 h. Diagnoses performed with the medium were confirmed by the BIOLOG and the fatty-acid profile methods. The diagnostic medium was used to determine the spatial distribution of colonized blossoms in the orchards and it was found that E. amylovora may be distributed both in clusters and at random. These findings were used in the development of a statistical model for sampling blossoms in the orchard. The model determines the number of trees to be sampled in the orchard and the number of blossoms be taken from each tree, which would enable the true colonization incidence of blossoms in the orchard to be estimated at desired levels of accuracy and confidence. Parameters included in the model are: the total number of trees in the orchard (T), the number of trees to be sampled in the orchard (t), the number of blossoms to be sampled from each tree (n), the true colonization incidence of blossoms (pi), a coefficient of aggregation (rho), the required level of confidence (1 - alpha), and the required level of accuracy (L). Sensitivity analyses revealed that the parameter governing sample size is the required level of accuracy. Sampling of 20 blossoms from each of several hundred trees is required to achieve an accuracy of +/-1%, but only a few single trees are needed for an accuracy level of +/-10%. A sampling procedure then was developed, validated with an independent data set, and found to be accurate. It was concluded that sampling of pear blossoms and estimation of the incidence of blossom colonization by E. amylovora could improve fire blight management, but not in all cases.

Characterization of the Erwinia 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.

Use of a diagnostic medium forin situ determination of the response ofErwinia amylovora 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. Aχ2 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.