Rootstock-regulated gene expression patterns associated with fire blight resistance in apple.

BACKGROUND: Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. RESULTS: Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. CONCLUSIONS: Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

Use of Gibberellic Acid for Management of Bunch Rot on Chardonnay and Vignoles Grape.

Harvest bunch rot of wine grape, caused primarily by Botrytis cinerea, is a perennial problem limiting the productivity of eastern vineyards, especially on cultivars with compact clusters. The aim of the present study was to evaluate the effectiveness of gibberellic acid (GA) sprays at reducing the compactness of Chardonnay and Vignoles clusters and minimizing bunch rot. Applications of GA reduced the number of berries per centimeter and the incidence and severity of bunch rots in Vignoles and, to a lesser extent, in Chardonnay over three consecutive years; however, the magnitude of GA effects often depended on the timing and rate of application. Bloom GA applications were more effective (P < 0.001) at reducing compactness and bunch rots than prebloom applications. Significantly, negative effects of GA applications on yield were negligible based on data from 4 years of trials on single vines and 2 years of data on 24-vine plots of Vignoles, provided the rates did not exceed 25 ppm. Regression analysis showed that berries per centimeter accounted for between 89 and 94% of variation in the incidence of Botrytis rot on Vignoles. On Chardonnay, compactness accounted for 53% of the variation in incidence, and the estimated compactness level at which no bunch rot would occur was 4.40 ± 1.05 (mean ± standard error) berries per centimeter. The relationship between cluster compactness and spray coverage of berries was also investigated in two separate experiments. Spray coverage of individual berries decreased linearly as cluster compactness increased within the range tested (3 to 18 berries per centimeter). Cluster compactness accounted for two-thirds of the variation in individual berry coverage, and coverage was reduced by 40 to 50% for clusters with about 18 berries per centimeter. These results strongly support the use of GA in integrated management of bunch rot on Vignoles and Chardonnay in eastern U.S. vineyards.

Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings.

The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, the most destructive bacterial disease of rosaceous plants, including apple and pear. Here, we compared the virulence levels of six E. amylovora strains (Ea273, CFBP1367, Ea581a, E2002a, E4001a, and HKN06P1) on apple trees and seedlings. The strains produced a range of disease severity, with HKN06P1 producing the greatest disease severity in every assay. We then compared virulence characteristic expression among the six strains, including growth rates in immature apple fruit, amylovoran production, levansucrase activity, biofilm formation, carbohydrate utilization, hypersensitive cell death elicitation in tobacco leaves, and protein secretion profiles. Multiple regression analysis indicated that three of the virulence characteristics (amylovoran production, biofilm formation, and growth in immature apple fruit) accounted for >70% of the variation in disease severity on apple seedlings. Furthermore, in greenhouse-grown 'Gala' trees, >75% of the variation in disease severity was accounted for by five of the virulence characteristics: amylovoran production, biofilm formation, growth in immature apple fruit, hypersensitive cell death elicitation, and sorbitol utilization. This study demonstrates that virulence factor expression levels account for differences in disease severity caused by wild isolates of E. amylovora on apple trees.

Diversity and biogeography of sooty blotch and flyspeck fungi on apple in the eastern and midwestern United States.

Sooty blotch and flyspeck (SBFS) fungi on apple fruit were sampled from nine orchards in four midwestern U.S. states during 2000 and 30 orchards in 10 eastern U.S. states during 2005 in order to estimate taxonomic diversity and discern patterns of geographic distribution. Forty apple fruit per orchard were arbitrarily sampled and colonies of each mycelial phenotype were counted on each apple. Representative colonies were isolated, cultures were purified, and DNA was extracted. For representative isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions of ribosomal DNA were amplified and sequenced. In total, 60 SBFS putative species were identified based on ITS sequences and morphological characteristics; 30 of these were discovered in the 2005 survey. Modified Koch's postulates were fulfilled for all 60 species in an Iowa orchard; colonies resulting from inoculation of apple fruit were matched to the original isolates on the basis of mycelial type and ITS sequence. Parsimony analysis for LSU sequences from both surveys revealed that 58 putative SBFS species were members of the Dothideomycetes, 52 were members of the Capnodiales, and 36 were members of the Mycosphaerellaceae. The number of SBFS species per orchard varied from 2 to 15. Number of SBFS species and values of the Margalef and Shannon indexes were significantly (P < 0.05) lower in 21 orchards that had received conventional fungicide sprays during the fruit maturation period than in 14 unsprayed orchards. Several SBFS species, including Schizothyrium pomi, Peltaster fructicola, and Pseudocercosporella sp. RH1, were nearly ubiquitous, whereas other species, such as Stomiopeltis sp. RS5.2, Phialophora sessilis, and Geastrumia polystigmatis, were found only within restricted geographic regions. The results document that the SBFS complex is far more taxonomically diverse than previously recognized and provide strong evidence that SBFS species differ in geographic distribution. To achieve more efficient management of SBFS, it may be necessary to understand the environmental biology of key SBFS species in each geographic region.

Relationship Between Cluster Compactness and Bunch Rot in Vignoles Grapes.

The impact of cluster compactness and debris retention on harvest bunch rot of Vitis interspecific hybrid 'Vignoles' was investigated between 2001 and 2005 near Lake Erie, in Pennsylvania. Cluster compactness was characterized subjectively based on the OIV code 204 standard and objectively by determining the number of berries per centimeter of rachis. In 2001, 2002, and 2004, the median number of berries per centimeter for loose clusters was 6.3, 7.0, and 6.4 compared with 10.2, 12.7, and 12.4 for the compact clusters, respectively. Kolmogorov-Smirnoff and the Mann-Whitney U two-samples tests confirmed that the distribution of the berries per centimeter was significantly (90 ≤ χ2 ≤ 184.3; P < 0.0001) different between the two subjective compactness categories. Cluster compactness was strongly correlated with bunch rot incidence (χ2 = 73.1 and 62.2 for 2001 and 2002, respectively; P < 0.0001), whereby disease incidence was higher in compact than in loose clusters. Logistic regression analysis indicated that every additional berry per centimeter unit of compactness almost doubled the likelihood of a cluster becoming infected with bunch rot (odds ratio = 1.828, 95% confidence interval [CI] = 1.392 to 2.399 in 2001 and odds ratio = 1.705, 95% CI = 1.394 to 2.085 in 2002). In 2004, bunch rot severity in compact clusters was nearly four times that of loose clusters. Linear regression analysis revealed that berries per centimeter accounted for >89% of the variation in bunch rot severity (R2 = 0.893, P < 0.0001, n = 30) and >74% in cluster weight (R2 = 0.745, P < 0.0001, n = 30). Accumulations of dehiscent floral debris contributed to greater bunch rot severity, and the effect was more pronounced in compact clusters than in loose clusters. Removal of basal leaves at trace bloom reduced berries per centimeter by 13% in 2004 and >25% in 2005, with corresponding reductions in bunch rot severity of 60% in 2004 and 62.5 to 82% in 2005. These results indicate that berries per centimeter is a good indicator of cluster compactness in Vignoles, and that practices that reduce cluster tightness would be effective in an integrated program for control of bunch rot on this cultivar.

Rootstock effects on gene expression patterns in apple tree scions.

Like many fruit trees, apple trees (Malus pumila) do not reproduce true-to-type from seed. Desirable cultivars are clonally propagated by grafting onto rootstocks that can alter the characteristics of the scion. For example, the M.7 EMLA rootstock is semi-dwarfing and reduces the susceptibility of the scion to Erwinia amylovora, the causal agent of fire blight disease. In contrast, the M.9 T337 rootstock is dwarfing and does not alter fire blight susceptibility of the scion. This study represents a comprehensive comparison of gene expression patterns in scions of the 'Gala' apple cultivar grafted to either M.7 EMLA or M.9 T337. Expression was determined by cDNA-AFLP coupled with silver staining of the gels. Scions grafted to the M.9 T337 rootstock showed higher expression of a number of photosynthesis-related, transcription/translation-related, and cell division-related genes, while scions grafted to the M.7 EMLA rootstock showed increased stress-related gene expression. The observed differences in gene expression showed a remarkable correlation with physiological differences between the two graft combinations. The roles that the differentially expressed genes might play in tree stature, stress tolerance, photosynthetic activity, fire blight resistance, and other differences conferred by the two rootstocks are discussed.