Fire Blight Resistance in Wild Accessions of Malus sieversii.

Fire blight (Erwinia amylovora) is a devastating bacterial disease in apple that results in severe economic losses. Epidemics are becoming more common as susceptible cultivars and rootstocks are being planted, and control is becoming more difficult as antibiotic-resistant strains develop. Resistant germplasm currently being utilized by breeding programs tend to have small fruit size and poor flavor characteristics. Malus sieversii, a progenitor species of domestic apple, is notable for its relatively large, palatable fruit and some accessions have been reported to be resistant to fire blight. In this study, nearly 200 accessions of M. sieversii and appropriate controls were inoculated with E. amylovora in both Washington and West Virginia to identify fire blight resistant accessions. Twelve accessions were identified with resistance comparable to highly resistant and resistant controls. Several accessions exhibited a unique resistance response, not previously reported in domestic apple (M. × domestica), characterized by low incidence of infection but high severity once infection was initiated. Several of these M. sieversii accessions will be used as parents in future crosses in the Washington State University apple breeding program.

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus 'Robusta 5' accessions.

BACKGROUND: Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus 'Robusta 5'. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. RESULTS: When several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with 'Robusta 5' as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand 'Malling 9' X 'Robusta 5' population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein (MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German 'Idared' X 'Robusta 5' population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene (HSP90). In the US 'Otawa3' X 'Robusta5' population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations. CONCLUSIONS: The results suggest that the upper region of 'Robusta 5' linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

Stable expression and phenotypic impact of attacin E transgene in orchard grown apple trees over a 12 year period.

BACKGROUND: Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health risk of the modified plants, transgene stability over a prolonged period of time and the effect of the gene on tree and fruit characteristics. We studied the stability of expression and the effect on resistance to the fire blight disease of the lytic protein gene, attacin E, in the apple cultivar 'Galaxy' grown in the field for 12 years. RESULTS: Using Southern and western blot analysis, we compared transgene copy number and observed stability of expression of this gene in the leaves and fruit in several transformed lines during a 12 year period. No silenced transgenic plant was detected. Also the expression of this gene resulted in an increase in resistance to fire blight throughout 12 years of orchard trial and did not affect fruit shape, size, acidity, firmness, weight or sugar level, tree morphology, leaf shape or flower morphology or color compared to the control. CONCLUSION: Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs. This report shows that it is possible to improve a desirable trait in apple, such as the resistance to a pathogen, through genetic engineering, without adverse alteration of fruit characteristics and tree shape.

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus x domestica) with Erwinia amylovora.

BACKGROUND: The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceae species, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora. RESULTS: cDNA were isolated from M.26 (susceptible) and G.41 (resistant) apple tissues collected 2 h and 48 h after challenge with a virulent E. amylovora strain or mock (buffer) inoculated. To identify differentially expressed transcripts, electrophoretic banding patterns were obtained from cDNAs. In the AFLP experiments, M.26 and G.41 showed different patterns of expression, including genes specifically induced, not induced, or repressed by E. amylovora. In total, 190 ESTs differentially expressed between M.26 and G.41 were identified using 42 pairs of AFLP primers. cDNA-AFLP analysis of global EST expression in a resistant and a susceptible apple genotype identified different major classes of genes. EST sequencing data showed that genes linked to resistance, encoding proteins involved in recognition, signaling, defense and apoptosis, were modulated by E. amylovora in its host plant. The expression time course of some of these ESTs selected via a bioinformatic analysis has been characterized. CONCLUSION: These data are being used to develop hypotheses of resistance or susceptibility mechanisms in Malus to E. amylovora and provide an initial categorization of genes possibly involved in recognition events, early signaling responses the subsequent development of resistance or susceptibility. These data also provided potential candidates for improving apple resistance to fire blight either by marker-assisted selection or genetic engineering.

Field Evaluation of Biological Control of Fire Blight in the Eastern United States.

The bacterial antagonists Pseudomonas fluorescens A506, Pantoea agglomerans C9-1, and Pantoea agglomerans E325 and preparations of Bacillus subtilis QST 713 containing bacterial endospores and lipopeptide metabolites were evaluated for efficacy in controlling fire blight in Michigan, New York, and Virginia. When examined individually, the biological control materials were not consistently effective in reducing blossom infection. The average reduction in blossom infection observed in experiments conducted between 2001 and 2007 was variable and ranged from 9.1 to 36.1%, while control with streptomycin was consistent and ranged from 59.0 to 67.3%. Incidence of blossom colonization by the bacterial antagonists was inconsistent, and <60% of stigmata had the antagonists present in 12 of 25 experiments. Consistent control of blossom infection was observed when the biological control materials were integrated into programs with streptomycin, resulting in a reduction of the number of streptomycin applications needed to yield similar levels of control. Our results indicate that the prospects for biological control of fire blight in the eastern United States are currently not high due to the variability in efficacy of existing biological control options.

Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease.

The Vf locus, originating from the crabapple species Malus floribunda 821, confers resistance to five races of the fungal pathogen Venturia inaequalis, the causal agent of apple scab disease. Previously, a cluster of four receptor-like genes, Vfa1, Vfa2, Vfa3, and Vfa4, was identified within the Vf locus. Because the amino-acid sequence of Vfa3 is truncated, it was deemed nonfunctional. In this study, each of the three full-length Vfa genes was introduced into a plant cloning vector, pCAMBIA2301, and used for Agrobacterium-mediated transformation of two apple cultivars, Galaxy and McIntosh, to assess functionality of these genes and to characterize their roles in resistance to V. inaequalis. Transformed apple lines carrying each of Vfa1, Vfa2, or Vfa4 were developed, analyzed for the presence of the transgene using polymerase chain reaction and Southern blotting, and assayed for resistance to apple scab following inoculation with V. inaequalis. Transformed lines expressing Vfa4 were found to be susceptible to apple scab, whereas those expressing either Vfa1 or Vfa2 exhibited partial resistance to apple scab. Based on Western blot analysis as well as microscopic analysis of plant resistance reactions, the roles of Vfa1 and Vfa2 in apple scab disease resistance response are discussed.

Isolation of Streptomycin-Resistant Isolates of Erwinia amylovora in New York.

Streptomycin is currently the only antibiotic registered for the control of fire blight, a devastating disease of apple (Malus), pear (Pyrus), and other rosaceous plants caused by the bacterium Erwinia amylovora. Resistance of E. amylovora to streptomycin was first identified in California pear orchards in 1971 and is currently endemic in many parts of the United States. The Northeast remains the only major U.S. apple-growing region without streptomycin-resistant isolates of E. amylovora. In 2002, during a routine survey for streptomycin resistance, isolates from two neighboring orchards in Wayne County, NY were found to be highly resistant to streptomycin at a concentration of 100 µg/ml. This constitutes the first authenticated report of streptomycin resistance in New York State. Infected trees were shipped at the same time from a single nursery in Michigan. Resistance was caused by the acquisition of the strA-strB gene pair, inserted into the ubiquitous nontransmissible E. amylovora plasmid pEA29. Previously, streptomycin-resistant E. amylovora populations from Michigan were described with a similar mechanism of resistance, although the strA-strB genes are not unique to Michigan. These findings illustrate how unintentional movement of nursery material could undermine efforts to prevent the spread of antibiotic-resistant E. amylovora.

Fire Blight Resistance of Budagovsky 9 Apple Rootstock.

Erwinia amylovora, the causal agent of fire blight, can cause a fatal infection of apple rootstocks known as rootstock blight. Budagovsky 9 (B.9) apple rootstock is reported to be highly susceptible when inoculated with E. amylovora, although results from multiple trials showed that B.9 is resistant to rootstock blight infection in field plantings. Conflicting results could stem from genetic variation in the B.9 population, appearing as phenotypic differences in rootstock material. However, genetic testing, using 23 microsatellite loci, confirmed the clonal uniformity of B.9 in commerce. Variation in growth habit between B.9 rootstocks originating from two nurseries also has been discounted as a source of disease resistance. Instead, results indicate a possible novel resistance phenotype in B.9 rootstock. B.9 rootstock was susceptible to leaf inoculation by E. amylovora, statistically similar to the susceptible rootstock Malling 9 (M.9). Conversely, inoculation assays targeting woody 4- to 5-year-old tissue revealed a high level of resistance in B.9, whereas M.9 remained susceptible. Although the mechanism by which B.9 gains resistance to E. amylovora is unknown, it is reminiscent of age-related resistance, due to an observed gain of resistance in woody rootstock tissue over succulent shoot tissue. Durable fire blight resistance correlated with tissue development could be a valuable tool for rootstock breeders.

Multiple models for Rosaceae genomics.

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.