Phenotypic Evaluation of Fire Blight Outbreak in the USDA Malus Collection

Fire blight, caused by pathogen Erwinia amylovora, is a major disease in Malus. Biological, chemical and cultural controls are efficient to manage fire blight, while rootstocks, and host resistance can limit damages. During the 2020 season a naturally occurring fire blight outbreak occurred in the United States Department of Agriculture (USDA) Malus collection, providing a unique opportunity to evaluate the diverse collection for fire blight susceptibility. The E. amylovora strain in the collection was identified as streptomycin resistant and characterized as CRISPR (clustered regularly interspaced short palindromic repeats) spacer array profile, 41:23:38. Fire blight severity was assessed using two approaches: (1) Average severity percentage, where the number of infected shoots was divided by the total number of shoots for the east and west facing sides of the tree; and (2) cut severity rating, where the trees were visually assessed after fire blight removal for amount of tree removed. Overall, 1142 trees of 41 Malus species were assessed for average severity and 2525 trees of 48 species were assessed for cut severity. A subset of 667 trees were for average severity in June and July to understand the disease progression. The species and trees presented here, can provide insight for future genetic fire blight resistance studies.

Molecular characterisation of apple accessions with respect to aminocyclopropane-1-carboxylic acid synthase gene (ACS1) polymorphism

The ARC apple gene bank collection was genotyped for the fruit expressed gene ACS1, in which a short-interspersed element (SINE) in the promoter is known, when homozygous, to correlate with the delayed ethylene production. Primers were designed amplifying products less than 500 bp and 224 cultivars of domestic apple were analysed, 169 not previously genotyped. Of these, 82 were aa (homozygous for the high ethylene allele at 202 bp), 73 were ab and 14 bb (homozygous for the low ethylene allele, with the SINE, at 339 bp). The difference between the allele sizes, 137 bp, observed in the current study is consistent with the indel of 138 bp originally described, but differs considerably from the indel of 166 bp reported in literature. In addition, 21 accessions of other Malus species were analysed. Only one, M. ‘Golden Hornet’, had the b allele, which suggests it may have been introgressed from M. pumila.

Comparative Proteomic Analysis Reveals Key Proteins Linked to the Accumulation of Soluble Sugars and Organic Acids in the Mature Fruits of the Wild Malus Species

Soluble sugars and organic acids are the main determinants of fruit organoleptic quality. To investigate the genes responsible for the soluble sugar and organic acid contents of apple fruits, a label-free proteomic analysis involving liquid chromatography (LC)-mass spectrometry (MS)/MS was conducted with the fruits of two Malus species, M. sargentii and M. niedzwetzkyana, which exhibit significant differences in soluble sugar and organic acid contents. A total of 13,036 unique peptides and 1,079 differentially-expressed proteins were identified. To verify the LC-MS/MS results, five candidate proteins were further analyzed by parallel reaction monitoring. The results were consistent with the LC-MS/MS data, which confirmed the reliability of the LC-MS/MS analysis. The functional annotation of the differentially-expressed proteins, based on the gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, revealed that they were mainly related to biological processes and cellular components. Additionally, the main enriched KEGG pathways were related to metabolic processes. Moreover, 31 proteins involved in soluble sugar metabolism, organic acid metabolism, and H+-transport were identified. The results of this study may be useful for the comprehensive characterization of the complex mechanism regulating apple fruit-soluble sugar and organic acid contents.

The Artificial Promoter rMdAG2I Confers Flower-specific Activity in Malus

Genetic modifications of floral organs are important in the breeding of Malus species. Flower-specific promoters can be used to improve floral organs specifically, without affecting vegetative organs, and therefore developing such promoters is highly desirable. Here, we characterized two paralogs of the Arabidopsis thaliana gene AGAMOUS (AG) from Malus domestica (apple): MdAG1 and MdAG2. We then isolated the second-intron sequences for both genes, and created four artificial promoters by fusing each intron sequence to a minimal 35S promoter sequence in both the forward and reverse directions. When transferred into tobacco (Nicotiana benthamiana) by Agrobacterium tumefaciens-mediated stable transformation, one promoter, rMdAG2I, exhibited activity specifically in flowers, whereas the other three also showed detectable activity in vegetative organs. A test of the four promoters' activities in the ornamental species Malus micromalus by Agrobacterium-mediated transient transformation showed that, as in tobacco, only rMdAG2I exhibited a flower-specific expression pattern. Through particle bombardment transformation, we demonstrated that rMdAG2I also had flower-specific activity in the apple cultivar ‘Golden Delicious’. The flower-specific promoter rMdAG2I, derived from M. domestica, thus has great potential for use in improving the floral characteristics of ornamental plants, especially the Malus species.

Determination of Predominant Organic Acid Components in Malus Species: Correlation with Apple Domestication

Significant variation in organic acid components was detected in mature fruits of 101 apple accessions using high-performance liquid chromatography. The Malus species predominantly accumulated malic acid and citric acid, whereas wild fruits exhibited significantly higher levels of organic acid content than that in cultivated fruits. Differential accumulation patterns during fruit developmental stages was detected between malic acid and citric acid, thus suggesting a complex genetic regulation mechanism of organic acid metabolism in apple fruit. A highly positive correlation was detected between fruit total organic acid content with malic acid and citric acid content, thus suggesting that malic acid and citric acid are the principal determinants of apple fruit acidity. In contrast to malic acid, citric acid was predominantly detected in partial wild apples, while extremely low to undetectable concentrations of citric acid were observed in cultivated apple fruits; this is likely due to the genetic effects of parental characters. Our results provide vital information that could be useful for future studies on genetic analysis and improvement of organic acid accumulation in apple fruits.