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Kristin E. Neill, Ryan N. Contreras, Virginia O. Stockwell, and Hsuan Chen

Erwinia amylovora . Bacterial cells that overwinter in fire blight cankers likely serve as primary inoculum in the spring ( Khan et al., 2012 ). Stem cankers produce bacterial ooze that is then disseminated by insects, wind, and rain to flowers or wounds

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David M. Hunter and W. Gordon Bonn

The virulence of six strains of Erwinia amylovora used in combination for screening fire blight resistance of pear seedlings and advanced selections from the Harrow pear breeding program was evaluated by inoculating a standardized suspension (108 cfu/ml) of the six strains individually and in combination into actively growing shoot tips and measuring the lengths of the diseased shoots six weeks later. Three cultivars provided a range of resistance to fire blight: `Bartlett' was susceptible, HW-605 (`Seckel' × NJ-6) was moderately resistant, while `Kieffer' was resistant. On `Bartlett', one strain was consistently more virulent than the combination, while on HW-605, two strains were consistently more virulent than the combination. One strain was consistently less virulent than the combination on both `Bartlett' and HW-605. No strain was consistently more or less virulent than the combination when inoculated into `Kieffer'. Lesion lengths were greater in the susceptible cultivar `Bartlett' than in either HW-605 or `Kieffer'. These results suggest that a combination of strains of E. amylovora is appropriate for screening for fire blight resistance in pear genotypes.

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Alejandro Martínez-Bilbao, Amaya Ortiz-Barredo, Emilio Montesinos, and Jesús Murillo

Fire blight, caused by the bacterium Erwinia amylovora (Burr.) Winsl. et al., is one of the most important diseases of apple and many other economically important genera in Rosaceae, not only for the economical losses that it generates, but also

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Philip J. Stewart, John R. Clark, and Patrick Fenn

Resistance to fire blight in blackberry (Rubus subgenus Rubus Watson) was studied in both seedling populations and clonally propagated plants. Seedling populations resulted from a partial diallel crossing of seven cultivars (Apache, Arapaho, Chester Thornless, Illini Hardy, Navaho, Triple Crown, and Prime-Jim™). Clonal material evaluated included eleven cultivars (Apache, Arapaho, Chester Thornless, Chickasaw, Kiowa, Illini Hardy, Navaho, Ouachita, Shawnee, Triple Crown, and Prime-Jim) and six breeding selections. Inoculations were made by injection of suspensions of Erwinia amylovora in sterile water. Significant differences in resistance were found among genotypes; `Navaho' was the most susceptible, and `Kiowa' and a breeding selection A-2095, the most resistant. Seedling inoculations showed resistance to be quantitatively inherited and mostly additive, with an overall narrow-sense heritability of h2 = 0.32.

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Kisung Ko, John L. Norelli, Jean-Paul Reynoird, Herb S. Aldwinckle, and Susan K. Brown

Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.

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Q. Liu, S. Salih, J. Ingersoll, R. Meng, L. Owens, and F. Hammerschlag

Transgenic `Royal Gala' apple (Malus × domestica Borkh.) shoots were obtained by Agrobacterium-mediated gene transfer using the plasmid binary vector pGV-osm-AC with a T-DNA encoding a chimeric gene consisting of a secretory sequence from barley-amylase joined to the modified cecropin MB39 coding sequence. Shoots were placed under the control of a wound-inducible, osmotin promoter from tobacco. The integration of the cecropin MB39 gene into apple was confirmed by Southern blot analysis. The transformation efficiency was 1.5% when internodes from etiolated shoots were used as explants and 2% when leaf explants were used. Both non- and transgenic tetraploid plants were produced by treatment of leaf explants with colchicine at 25 mg·L-1, and polyploidy was confirmed by flow cytometry. Of the diploid transgenics, three of seven were significantly more resistant to Erwinia amylovora than the non-transgenic `Royal Gala' control. Also, in one instance, a tetraploid transgenic was significantly more resistant than the diploid shoot from which it was derived.

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Andrew C. Bell, Thomas G. Ranney, Thomas A. Eaker, and Turner B. Sutton

appreciated. The authors wish to thank Don Shadow, Ken Tilt, the Arnold Arboretum, and the Landscape Plant Development Center for providing plant material and Jay Norelli and Herb Aldwinckle who generously provided the isolate of Erwinia amylovora used for

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A.R. Biggs

Three apple (Malus domestics Borkh.) cultivars varying in susceptibility to Erwinia amylovora (Burr.) Winslow et al., the causal agent of tire blight, were inoculated at biweekly intervals during the growing season. Data were collected on percent infection, canker length, and canker margin quality (a reflection of the overwintering status of the infection). There was a significant cultivar × noculation date interaction, indicating that cultivars that are more susceptible to the pathogen are more likely to develop cankers with indeterminate margins. For `Cortland' and `Jonathan', but not `Delicious', there was a significant linear trend toward forming indeterminate cankers as inoculations were made later in the season. Cankers initiated earlier in the season were more likely to be determinate, which suggests that later-season infections on susceptible cultivars carry over inoculum to the following season. No specific switch-over period from determinate- to indeterminate-type cankers could be identified, and canker margin qualities changed gradually during the growing season. As expected, `Delicious' appeared resistant to tire blight in this study, based on percent infection, canker length, and canker margin type, whereas `Cortland' and `Jonathan' appeared moderately and highly susceptible, respectively. `Delicious' was more likely to form cankers with determinate margins, which suggests that cankers formed on this cultivar are less likely to produce inoculum in the spring than the other two cultivars.

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T. van der Zwet and R.L. Bell

During 1976-1980, three plant exploration trips were made throughout eastern Europe in search of native Pyrus germplasm. A total of 384 accessions (231 from Yugoslavia, 86 from Romania, 43 from Poland, and 12 each from Hungary and Czechoslovakia) were collected as budwood and propagated at the National Plant Germplasm Quarantine Center in Glenn Dale, Md. Following 8 years of exposure to the fire blight bacterium [Erwinia amylovora (Burr.) Winsl. et al.], 17.49” of the accessions remained uninfected, 11.2% rated resistant, 6.8% moderately resistant, and 64.6% blighted severely (26% to 100% of tree blighted). Some of the superior accessions have been released for use in the pear breeding program.

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Tom van der Zwet and Richard L. Bell

Of 133 Pyrus accessions (predominantly P. communis L.), collected in Central Europe and previously rated in the resistant U.S. Dept. of Agriculture (USDA) blight scores 10–6, only 77 (57.0%) remained in these scores after an additional 5 years of exposure to fire blight [Erwinia amylovora (Burr.) Winsl. et al.]. Of these, 24 originated from three states in former Yugoslavia. Following several years of severe blight epiphytotics, only 5 (10.4%) of 52 accessions released from quarantine since 1986 and planted at Appalachian Fruit Research Station scored 6 or above. All accessions were highly susceptible to artificial blossom inoculation, and only 10 accessions were at least moderately resistant to artificial shoot inoculations.